Immunoglobulins

ABSTRACT

The present invention relates to antibodies to NOGO, pharmaceutical formulations containing such antibodies and the use of such antibodies in the treatment and/or prophylaxis of neurological diseases/disorder.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is continuation of application Ser. No. 12/097,279,filed on Jun. 13, 2008, which is the US National Stage Application ofInternational Application No. PCT/EP2006/069737, filed Dec. 14, 2006,which claims priority from Great Britain Application No. 0525662.3,filed Dec. 16, 2005, the contents of all of which are hereinincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to immunoglobulins, particularlyantibodies that bind to NOGO and neutralise the activity thereof,polynucleotides encoding such antibodies, pharmaceutical formulationscontaining said antibodies and to the use of such antibodies in thetreatment and/or prophylaxis of neurological diseases. Other aspects,objects and advantages of the present invention will become apparentfrom the description below.

BACKGROUND OF THE INVENTION

Stroke is a major cause of death and disability in the Western World.There is no approved therapy for the treatment of stroke other thantissue plasminogen (t-PA) which has to be administered within 3 hours ofonset following a computer tomography (CT) scan to exclude haemorrhage.To date most therapeutic agents directed towards the treatment of acutestroke (i.e. neuroprotection), have predominantly involved targetingglutamate receptors and their down stream signalling pathways known tobe involved in acute cell death. However to date these strategies haveproved unsuccessful in clinical trials and are often associated withdose-limiting side effects (Hill & Hachinski, The Lancet, 352: (supplIII) 10-14 (1998)). Therefore there is a need for novel approachesdirected towards the amelioration of cell death following the cessationof blood flow. Neuroprotection is the ability of a treatment to preventor ameliorate neuronal cell loss in response to an insult or diseaseprocess. This may be achieved by targeting the neurons directly orindirectly by preventing glial (including oligodendrocyte) cell loss.

Following the onset of stroke, some degree of spontaneous functionalrecovery is observed in many patients, suggesting that the brain has the(albeit limited) ability to repair and/or remodel following injury.Agents that have the potential to enhance this recovery may thereforeallow intervention to be made much later (potentially days) followingthe onset of cerebral ischaemia. Agents which are able to offer bothacute neuroprotection and enhance functional recovery may providesignificant advantages over current potential neuroprotectivestrategies.

Alzheimer's disease (AD) is characterised by the presence of twodiagnostic features of pathology. These are amyloid plaques andneurofibrillary tangles composed of aggregated beta-amyloid peptide(Aβ40 and Aβ42) and hyperphosphorylated tau respectively (Dawbarn &Allen 2001 Neurobiology of Alzheimer's Disease OUP).

A comprehensive study has shown a strong link in patients betweenbeta-amyloid accumulation and cognitive decline (Naslund et al, JAMA,Mar. 22/29, 2000, Vol. 283, No; 12, page 1571-1577). This is consistentwith genetic and epidemiological studies that suggest that somemutations in APP and presenilin genes can predispose to early onset AD,which mutations also enhance the levels of Aβ40 and Aβ42 peptide,including the ratio thereof.

Cleavage of the type I transmembrane amyloid precursor protein (APP) bytwo distinct proteases designated beta- and gamma-secretase is necessaryfor the formation of beta-amyloid peptide. The molecular identity ofbeta-secretase as the aspartyl-protease Asp2/BACE1 has been confirmed(Hussain et al Mol. Cell. NeuroSci. 16, 609-619 (2000); Vassar et al,Science (1999), Oct. 22; 286 (5440):735-741). The nature ofgamma-secretase remains the source of some debate and is likely toconsist of a high molecular weight complex consisting of at least thefollowing proteins: presenilins, Aph1, Pen2 and nicastrin (reviewed inMedina & Dotti Cell Signalling 2003 15(9):829-41).

The processing of APP within the CNS is likely to occur within a numberof cell-types including neurons, oligodendrocytes, astrocytes andmicroglia. While the overall rate of APP processing in these cells willbe influenced by the relative level of expression of APP, BACE1/Asp2,presenilin-1 and -2, Aph1, Pen2 and nicastrin.

Furthermore, additional factors regulating the subcellular location ofAPP can also influence its processing as shown by the finding thatmutation of the YENP motif in the APP cytoplasmic domain which blocksits endocytosis reduces beta-amyloid production (Perez et al 1999 J BiolChem 274 (27) 18851-6). Retention of the APP-beta-CTF in the ER by theaddition of the KKQN retention motif is sufficient to reduce amyloidproduction in transfected cells (Maltese et al 2001 J Biol Chem 276 (23)20267-20279). Conversely, elevation of endocytosis, by overexpression ofRab5 is sufficient to elevate amyloid secretion from transfected cells(Grbovic et al 2003 J Biol Chem 278 (33) 31261-31268).

Consistent with these findings further studies have shown that reductionof cellular cholesterol levels (a well known risk factor for AD) reducedbeta-amyloid formation. This change was dependent on altered endocytosisas demonstrated by the use of the dominant negative dynamin mutants(K44A) and overexpression of the Rab5 GTPase activating protein RN-Tre(Ehehalt et al 2003 J Cell Biol 160 (1) 113-123).

Cholesterol rich microdomains or rafts are also an important cellularsite of beta-amyloid production and APP, BACE1 and components of thegamma-secretase complex have all been shown to transiently reside withinrafts. Antibody cross-linking of APP and BACE1 towards cholesterol richrafts was able to elevate beta-amyloid production (Ehehalt et al 2003 JCell Biol 160 (1) 113-123). Expression of GPI-anchored BACE1, which isexclusively targeted to lipid rafts, is similarly able to elevate APPcleavage and beta-amyloid production (Cordy et al 2003 PNAS 100(20)11735-11740).

The mechanisms underlying functional recovery after a stroke or otherneurodamaging event or disease, are currently unknown. The sprouting ofinjured or non-injured axons has been proposed as one possiblemechanism. However, although in vivo studies have shown that treatmentof spinal cord injury or stroke with neurotrophic factors results inenhanced functional recovery and a degree of axonal sprouting, these donot prove a direct link between the degree of axonal sprouting andextent of functional recovery (Jakeman, et al. 1998, Exp. Neurol. 154:170-184, Kawamata et al. 1997, Proc Natl Acad. Sci. USA., 94:8179-8184,Ribotta, et al. 2000, J Neurosci. 20: 5144-5152). Furthermore, axonalsprouting requires a viable neuron. In diseases such as stroke which isassociated with extensive cell death, enhancement of functional recoveryoffered by a given agent post stroke may therefore be through mechanismsother than axonal sprouting such as differentiation of endogenous stemcells, activation of redundant pathways, changes in receptordistribution or excitability of neurons or glia (Fawcett & Asher, 1999,Brain Res. Bulletin, 49: 377-391, Horner & Gage, 2000, Nature 407963-970).

The limited ability of the central nervous system (CNS) to repairfollowing injury is thought in part to be due to molecules within theCNS environment that have an inhibitory effect on axonal sprouting(neurite outgrowth). CNS myelin is thought to contain inhibitorymolecules (Schwab M E and Caroni P (1988) J. Neurosci. 8, 2381-2193).Two myelin proteins, myelin-associated glycoprotein (MAG) and NOGO havebeen cloned and identified as putative inhibitors of neurite outgrowth(Sato S. et al (1989) Biochem. Biophys. Res. Comm. 163, 1473-1480;McKerracher L et al (1994) Neuron 13, 805-811; Mukhopadhyay G et al(1994) Neuron 13, 757-767; Torigoe K and Lundborg G (1997) Exp.Neurology 150, 254-262; Schafer et al (1996) Neuron 16, 1107-1113;WO9522344; WO9701352; Prinjha R et al (2000) Nature 403, 383-384; Chen MS et al (2000) Nature 403, 434-439; GrandPre T et al (2000) Nature 403,439-444; US005250414A; WO200005364A1; WO0031235).

Three forms of human NOGO have been identified: NOGO-A having 1192 aminoacid residues (GenBank accession no. AJ251383); NOGO-B, a splice variantwhich lacks residues 186 to 1004 in the putative extracellular domain(GenBank accession no. AJ251384) and a shorter splice variant, NOGO-C,which also lacks residues 186 to 1004 and also has smaller, alternativeamino terminal domain (GenBank accession no. AJ251385) (Prinjha et al(2000) supra).

Inhibition of the CNS inhibitory proteins such as NOGO may provide atherapeutic means to ameliorate neuronal damage and promote neuronalrepair and growth thereby potentially assisting recovery from neuronalinjury such as that sustained in stroke. Examples of such NOGOinhibitors may include small molecules, peptides and antibodies.

It has been reported that a murine monoclonal antibody, IN-1, that wasraised against NI-220/250, a myelin protein which is a potent inhibitorof neurite growth (and subsequently shown to be fragment of NOGO-A),promotes axonal regeneration (Caroni, P and Schwab, M E (1988) Neuron 185-96; Schnell, L and Schwab, M E (1990) Nature 343 269-272; Bregman, BS et al (1995) Nature 378 498-501 and Thallmair, M et al (1998) NatureNeuroscience 1 124-131). It has also been reported that NOGO-A is theantigen for IN-1 (Chen et al (2000) Nature 403 434-439). Administrationof IN-1 Fab fragment or humanised IN-1 to rats that have undergonespinal cord transection, enhanced recovery (Fiedler, M et al (2002)Protein Eng 15 931-941; Brosamle, C et al (2000) J. Neuroscience 208061-8068).

Monoclonal antibodies which bind to NOGO are described in WO 04/052932and WO2005028508. WO 04/052932 discloses a murine antibody 11C7 whichbinds to certain forms of human NOGO with high affinity.

Patent application WO05/061544 also discloses high affinity monoclonalantibodies, including a murine monoclonal antibody 2A10, and generallydiscloses humanised variants thereof, for example H1L11 (the sequencesfor the H1 and L11 are provided in SEQ ID NOs. 33 and 34 respectively(VH or VL sequences only)). The antibodies disclosed bind to humanNOGO-A with high affinity. The murine 2A10 antibody (and CDR-graftedhumanised variants thereof) are characterised by the followingcomplementarity determining region (CDR) sequences (as determined usingthe Kabat methodology (Kabat et al. (1991) “Sequences of proteins ofimmunological interest”; Fifth Edition; US Department of Health andHuman Services; NIH publication No 91-3242)) within their light andheavy chain variable regions:

TABLE 1 Antibody 2A10 light chain CDRs CDR Sequence L1RSSKSLLYKDGKTYLN (SEQ ID NO: 4) L2 LMSTRAS (SEQ ID NO: 5) L3QQLVEYPLT (SEQ ID NO: 6)

TABLE 2 Antibody 2A10 heavy chain CDRs CDR Sequence H1SYWMH (SEQ ID NO: 1) H2 NINPSNGGTNYNEKFKS (SEQ ID NO: 2) H3GQGY (SEQ ID NO: 3)

WO05/061544 further discloses “analogues” of the antibodies thatcomprise the CDRs of Tables 1 and 2 above, such “analogues” the havesame antigen binding specificity and/or neutralizing ability as thedonor antibody from which they were derived.

Despite the art providing high affinity anti-NOGO antibodies, it remainsa highly desirable goal to isolate and develop alternative, or improved,therapeutically useful monoclonal antibodies that bind and inhibit theactivity of human NOGO.

The process of neurodegeneration underlies many neurologicaldiseases/disorders including, but not limited to, acute diseases such asstroke (ischemic or haemorrhagic), traumatic brain injury and spinalcord injury as well as chronic diseases including Alzheimer's disease,fronto-temporal dementias (tauopathies), peripheral neuropathy,Parkinson's disease, Creutzfeldt-Jakob disease (CJD), Schizophrenia,amyotrophic lateral sclerosis (ALS), multiple sclerosis, Huntington'sdisease, multiple sclerosis and inclusion body myositis. Consequentlythe anti-NOGO monoclonal antibodies, and the like, of the presentinvention may be useful in the treatment of these diseases/disorders.Antibodies for the treatment of the above mentioned disease/disordersare provided by the present invention and described in detail below.

BRIEF SUMMARY OF THE INVENTION

The invention provides specific heavy chain variable regions, andantibodies or fragments thereof comprising the said specific heavy chainvariable regions and a light chain variable region that allows, whenpaired with the heavy chain variable regions, the Fv dimer to bind humanNOGO-A with high affinity, and thereby neutralise the activity of humanNOGO-A.

The heavy chain variable regions of the present invention may beformatted, together with light chain variable regions to allow bindingto human NOGO-A, in the conventional immunoglobulin manner (for example,human IgG, IgA, IgM etc.) or in any other fragment thereof or“antibody-like” format that binds to human NOGO-A (for example, singlechain Fv, diabodies, Tandabs™ etc (for a summary of alternative“antibody” formats see Holliger and Hudson, Nature Biotechnology, 2005,Vol 23, No. 9, 1126-1136)).

A heavy chain variable region comprising a third CDR consistingessentially of the amino acid residues GQGY wherein the CDR contains atleast one substitution within the GQGY core sequence, the substitutionsbeing selected from the following substitutions: where the G in thefirst position is replaced by R, I, W or M; the Q in the second positionis replaced by D, I, A, L, V or S; the G in the third position isreplaced by W, N, Y, S, L or F; and the Y in the fourth position isreplaced by W.

In another embodiment, the third heavy chain CDR (CDR H3) only containsone substitutions to yield the following CDR H3: RQGY (SEQ ID NO.75),IQGY (SEQ ID NO.76), MQGY (SEQ ID NO.45), GDGY (SEQ ID NO.77), GIGY (SEQID NO.78), GSGY (SEQ ID NO.79), GQNY (SEQ ID NO.80), GQYY (SEQ IDNO.81), GQSY (SEQ ID NO.62), GQLY (SEQ ID NO.82), GQFY (SEQ ID NO.83),GQGW (SEQ ID NO.84), WQGY(SEQ ID NO.86), GAGY (SEQ ID NO.87), GLGY (SEQID NO.88), GVGY (SEQ ID NO.89), GQWY (SEQ ID NO.90).

In another embodiment, the heavy chain variable regions above furthercontain the other CDRs listed in Table 2, i.e. CDR H1 (SEQ ID NO. 1) andCDR H2 (SEQ ID NO.2).

The antibodies of the present invention, or fragments thereof, retainthe human NOGO binding activity of antibodies that comprise the CDR H3:GQGY, in terms of their activity as measured in ELISA and Biacoreexperiments, and in some cases the activity in these experiments isincreased.

Human or Humanised Heavy Chain Variable Regions Containing G95M(Substitution Numbering by Kabat)

In one embodiment of the present invention, the heavy chain variableregions of the present invention comprise the CDRs defined in Table 3(as defined by Kabat):

TABLE 3 CDR Sequence H1 SYWMH (SEQ ID NO: 1) H2NINPSNGGTNYNEKFKS (SEQ ID NO: 2) H3 MQGY (SEQ ID NO: 45)

In one embodiment of the present invention there is provided a human orhumanised heavy chain variable region comprising each of the CDRs listedin Table 3. In another embodiment of the present invention there isprovided a humanised heavy chain variable region comprising the CDRslisted in Table 3 within the larger sequence of a human heavy chainvariable region. In yet another embodiment the humanised heavy chainvariable region comprises the CDRs listed in Table 3 within an acceptorantibody framework having greater than 40% identity in the frameworkregions, or greater than 50%, or greater than 60%, or greater than 65%identity to the murine 2A10 donor antibody heavy chain variable region(SEQ ID NO.7).

When the CDRs of Table 3 are all used, in one embodiment the heavy chainvariable region sequence is sequence H98 provided as SEQ ID NO. 66 (H98VH is the equivalent of H1 VH (SEQ ID NO.33) differing only in that theCDR H3 is MQGY in H98 instead of GQGY as found in H1).

In one aspect of the present invention the antibodies comprise a heavychain variable region having the amino acid sequence of SEQ ID NO. 66(H98 variable region) further comprising a number of substitutions atone or more of positions 38, 40, 48, 67, 68, 70, 72, 74, and 79; whereineach substituted amino acid residue is replaced with the amino acidresidue at the equivalent position in SEQ ID NO 7 (the heavy chainvariable region of the donor antibody 2A10) and the number ofsubstitutions is between 1 and 9. In other embodiments the number ofsubstitutions is 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8 or 9.

In this context the substitutions that are described are equivalent inconcept to “back-mutations” where the human framework amino acidresidues in specific positions within the H98 sequence are back-mutatedto the amino acid residues in the equivalent position within the 2A10donor antibody sequence.

Unless specifically stated otherwise to the contrary herein, when anumerical position of an amino acid residue found within a specificsequence is mentioned in this document, for example “position 12”, it isintended that the skilled reader assigns the first amino acid in thesequence the position “1” and counts from position one and identifiesthe amino acid which is in the desired position, in this example thetwelfth amino acid residue in the sequence. The skilled reader willnotice that this numbering system does not correspond with the Kabatnumbering system which is often used to define amino acid positionswithin antibody sequences.

For optimal binding affinity, it was found for the humanisation of themouse antibody 2A10 (the VH for which is SEQ ID NO. 7) that the pair ofamino acid residues in positions 48 and 68, should be I and Arespectively (as they exist in 2A10) or M and V respectively (as theyexist in H98). It is expected that the above finding is also ofrelevance to the humanisation of the G95M variant of 2A10.

The following table includes details of three different heavy chainvariable (VH) regions which may form part of the antibodies of thepresent invention. Each of the disclosed VH is based on the H98 VH (SEQID NO. 66) further comprising the substitutions mentioned in the table(Table 4) where the H98 residue at the relevant position is substitutedwith the 2A10 residue at that position (in the table, “−” means thatthere is no substitution in that position, and so the residue remains asin the sequence of H98):

TABLE 4 Numerical Residue No. 38 40 48 67 68 70 72 74 79 Kabat No. 38 4048 66 67 69 71 73 78 2A10 New VH K R I K A L V K A (SEQ ID H98 NO. X) RA M R V M R T V H26 (47) — — I — A — — — A H27 (48) K R I K A L V K AH28 (49) — — I K A — — — A

In one embodiment of the present invention, therefore, the heavy chainvariable regions (VH) of the present invention are H26 VH (SEQ ID NO.47), H27 VH (SEQ ID NO. 48) and H28 VH (SEQ ID NO. 49)

SEQ ID 47: VH humanised construct H26QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGNINPSNGGTNYNEKFKSRATMTRDTSTSTAYMELSSLRSEDTAVYYC ELMQGYWGQGTLVTVSSSEQ ID 48: VH humanised construct H27QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVKQRPGQGLEWIGNINPSNGGTNYNEKFKSKATLTVDKSTSTAYMELSSLRSEDTAVYYC ELMQGYWGQGTLVTVSSSEQ ID 49: VH humanised construct H28QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGNINPSNGGTNYNEKFKSKATMTRDTSTSTAYMELSSLRSEDTAVYYC ELMQGYWGQGTLVTVSS

Human or Humanised Heavy Chain Variable Regions Including G101S(Substitution Numbering by Kabat)

In another embodiment of the present invention there is provided a humanor humanised heavy chain variable region which comprises CDRs defined inTable 5:

TABLE 5 CDR According to Kabat H1 SYWMH (SEQ ID NO: 1) H2NINPSNGGTNYNEKFKS (SEQ ID NO: 2) H3 GQSY (SEQ ID NO: 62)

In one embodiment the CDRs of Table 5 are incorporated within a humanheavy chain variable region sequence. In another embodiment thehumanised heavy chain variable region comprises the CDRs listed in Table5 within an acceptor antibody framework having greater than 40% identityin the framework regions, or greater than 50%, or greater than 60%, orgreater than 65% identity to the murine 2A10 donor antibody heavy chainvariable region (SEQ ID NO.7).

In another embodiment the CDRs of Table 5 are inserted into a humanheavy chain variable region to give the following sequence (H99):

(SEQ ID 61: 2A10 VH humanised construct H99)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGNINPSNGGTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYC ELGQSYWGQGTLVTVSS.

In other embodiments, further back mutations are added to the H99 VHsequence in any one of positions (denoted by numerical residue position)38, 40. 48, 67, 68, 70, 72, 74 or 79; wherein each substituted aminoacid residue is replaced with the amino acid residue at the equivalentposition in SEQ ID NO 7 (the heavy chain variable region of the donorantibody 2A10) and the number of substitutions is between 1 and 9. Inother embodiments the number of substitutions is 1, or 2, or 3, or 4, or5, or 6, or 7, or 8 or 9. H99 VH is the equivalent of H1 VH (SEQ IDNO.33) differing only in that the CDR H3 is GQSY in H99 instead of GQGYas found in H1.

For optimal binding affinity, it was found for the humanisation of themouse antibody 2A10 (the VH for which is SEQ ID NO. 7) that the pair ofamino acid residues in positions 48 and 68, should be I and Arespectively (as they exist in 2A10) or M and V respectively (as theyexist in H98). It is expected that the above finding is also ofrelevance to the humanisation of the G95M variant of 2A10.

In one embodiment the back mutations are located in the positionsindicated in Table 6 below where the H99 residue at the relevantposition is substituted with the 2A10 residue at that position (in thetable, “−” means that there is no substitution in that position, and sothe residue remains as in the sequence of H1):

Table 6,

TABLE 6 Numerical Residue No. 38 40 48 67 68 70 72 74 79 Kabat No. 38 4048 66 67 69 71 73 78 2A10 New VH K R I K A L V K A (SEQ ID H99 NO. X) RA M R V M R T V H100 (63) — — I — A — — — A H101 (64) K R I K A L V K AH102 (65) — — I K A — — — AAntibodies or Fragments that Comprise the Human or Humanised Heavy ChainVariable Regions and Light Chain Variable Regions

The VH constructs of the present invention may be paired with a lightchain to form a human NOGO-A binding unit (Fv) in any format, includinga conventional IgG antibody format having full length (FL) variable andconstant domain heavy chain sequences. Examples of full length (FL) IgG1heavy chain sequences comprising the VH constructs of the presentinvention and inactivating mutations in positions 235 and 237 (EU Indexnumbering) to render the antibody non-lytic are SEQ ID NOs 53, 54 and55.

SEQ ID 53: Heavy chain humanised construct H26MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGNINPSNGGTNYNEKFKSRATMTRDTSTSTAYMELSSLRSEDTAVYYCELMQGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK.SEQ ID 54: Heavy chain humanised construct H27MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVKQRPGQGLEWIGNINPSNGGTNYNEKFKSKATLTVDKSTSTAYMELSSLRSEDTAVYYCELMQGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGKSEQ ID 55: Heavy chain humanised construct H28MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGNINPSNGGTNYNEKFKSKATMTRDTSTSTAYMELSSLRSEDTAVYYCELMQGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK

The light chain variable region sequence that forms an Fv with the heavychain variable region sequences of the present invention may be anysequence that allows the Fv to bind to Human NOGO-A.

In one embodiment of the present invention the light chain variableregion is the 2A10 light chain (see WO 05/061544), the light chainvariable region of which is provided herein as SEQ ID NO. 8 or humanisedvariants thereof. Humanised variants of the 2A10 light chain preferablycontain all of the light chain variable region CDRs that are describedin Table 1 grafted onto a human light chain variable region acceptorframework. In one embodiment the humanised light chain variable regionsare L11 (SEQ ID NO.34), L13 (SEQ ID NO.13) or L16 (SEQ ID NO.14).Alternative light chain variable regions that are based on L13 and L16,which comprise specific substitutions in kabat positions 37 and/or 45,are provided in Table 7.

TABLE 7 SEQ Description ID NO. Sequence L100 (L13 + Q37R) 67DIVMTQSPLSLPVTLGQPASISCRSSKSLLYKDGKTYLNWFRQRPGQSPQLLIYLMSTRASGVPDRFSGGGSGTDFTLKISRVEA EDVGVYYCQQLVEYPLTFGQGTKLEIKL101 (L13 + Q45R) 68 DIVMTQSPLSLPVTLGQPASISCRSSKSLLYKDGKTYLNWFQQRPGQSPRLLIYLMSTRASGVPDRFSGGGSGTDFTLKISRVEAE DVGVYYCQQLVEYPLTFGQGTKLEIKL102 69 DIVMTQSPLSLPVTLGQPASISCRSSKSLLYKDGKTYLNWFRQ (L13 + Q37R/Q45R)RPGQSPRLLIYLMSTRASGVPDRFSGGGSGTDFTLKISRVEAE DVGVYYCQQLVEYPLTFGQGTKLEIKL103 (L16 + Q37R) 70 DIVMTQSPLSNPVTLGQPVSISCRSSKSLLYKDGKTYLNWFRQRPGQSPQLLIYLMSTRASGVPDRFSGGGSGTDFTLKISRVEA EDVGVYYCQQLVEYPLTFGQGTKLEIKL104 (L16 + Q45R) 71 DIVMTQSPLSNPVTLGQPVSISCRSSKSLLYKDGKTYLNWFLQRPGQSPRLLIYLMSTRASGVPDRFSGGGSGTDFTLKISRVEAE DVGVYYCQQLVEYPLTFGQGTKLEIKL105 72 DIVMTQSPLSNPVTLGQPVSISCRSSKSLLYKDGKTYLNWFRQ (L16 + Q37R/Q45R)RPGQSPRLLIYLMSTRASGVPDRFSGGGSGTDFTLKISRVEAE DVGVYYCQQLVEYPLTFGQGTKLEIK

In another embodiment the full length (FL) light chain sequences areL11FL (SEQ ID NO.36), L13FL (SEQ ID NO.17) or L16FL (SEQ ID NO.18).

In another embodiment the antibodies, fragments or functionalequivalents thereof comprise a VH sequence selected from H26, H27, H28,H100, H101 and H102; in combination with any one of the following VLsequences L11, L13, L16, L100, L101, L102, L103, L104, and L105. It isintended that all possible combinations of the listed heavy chainvariable regions and light chain variable regions be specificallydisclosed (e.g. H28L104 et. al.).

In particular embodiments the antibodies, fragments or functionalequivalents thereof comprise the following variable region pairs:

H27L16 (SEQ ID NO.48+SEQ ID NO.14)

H28L13 (SEQ ID NO.49+SEQ ID NO.13)

H28L16 (SEQ ID NO.49+SEQ ID NO.14)

In another embodiment the antibodies of the present invention comprisethe following full length sequences:

H27FL L16FL (SEQ ID NO. 54+SEQ ID NO.18)

H28FL L13FL (SEQ ID NO. 55+SEQ ID NO.17)

H28FL L16FL (SEQ ID NO. 55+SEQ ID NO.18)

In one embodiment the antibody of the present invention comprises H27L16(SEQ ID NO.48+SEQ ID NO.14), or is the full length antibody H28FL L16FL(SEQ ID NO. 55+SEQ ID NO.18).

In another embodiment, the antibody or fragment thereof binds to thesame human NOGO epitope as H28L16, or competes with the binding ofH28L16 to human NOGO, characterised in both instances in that thecompeting antibody, or fragment thereof, is not the murine antibody 2A10or a human or humanised variant thereof comprising a CDR H3 having thesequence GQGY (SEQ ID NO.3) or a sequence containing one amino acidsubstitution in the CDR H3.

In particular embodiments the antibodies, fragments or functionalequivalents thereof comprise the following variable region pairs:

H100L16 (SEQ ID NO.63+SEQ ID NO.14)

H101L13 (SEQ ID NO.64+SEQ ID NO.13)

H102L16 (SEQ ID NO.65+SEQ ID NO.14)

Epitope Mapping and Further Antibodies that Bind to the Same Epitope

In another embodiment there is provided an antibody, or fragmentthereof, that is capable of binding to human NOGO protein, or fragmentthereof such as a GST-NOGO-A56 protein (SEQ ID NO.32), in an ELISAassay, wherein the binding of the antibody, or fragment thereof, to thehuman NOGO protein, or fragment thereof, in the ELISA assay is reducedin the presence of a peptide having the following sequence VLPDIVMEAPLN(SEQ ID NO. 60), and is not reduced in the presence of an irrelevantpeptide, for instance a peptide from human NOGO that does not overlapwith SEQ ID NO.60 (such as SEQ ID NO. 85, YESIKHEPENPPPYEE),characterised in that the antibody or fragment thereof is not anantibody comprising a heavy chain variable domain having a CDR H3consisting of the amino acid residues GQGY or analogues thereof havingone amino acid substitution in the CDR H3. Alternatively the competingpeptide is TPSPVLPDIVMEAPLN (SEQ ID NO. 73) or VLPDIVMEAPLNSAVP (SEQ IDNO. 74). In addition, the antibody that binds to the same epitope as theantibodies, or fragments thereof, may be an antibody that does notcomprise all of the CDRs listed in Tables 1 and 2, or any antibody thatcomprises a set of CDRs that has 80% or greater homology to the CDRslisted in Tables 1 and 2 combined, or Tables 1 or 2 alone.

In another embodiment of the present invention there is provided anantibody or fragment thereof, that is capable of binding in an ELISAassay to a region of human NOGO protein consisting of the polypeptidesequence of VLPDIVMEAPLN (SEQ ID NO. 60), characterised in that theantibody, or fragment thereof is not an antibody comprising a variableheavy domain having CDR H3 consisting of the amino acid residues GQGY oranalogues thereof having one amino acid substitution in the CDR H3.Alternatively the antibody or fragment thereof is capable of binding toTPSPVLPDIVMEAPLN (SEQ ID NO. 73) or VLPDIVMEAPLNSAVP (SEQ ID NO. 74). Inaddition, the antibody that binds to the same epitope as the antibodies,or fragments thereof, may be an antibody that does not comprise all ofthe CDRs listed in Tables 1 and 2, or any antibody that comprises a setof CDRs that has 80% or greater homology to the CDRs listed in Tables 1and 2 combined, or Tables 1 or 2 alone.

In another embodiment of the present invention there is provided amethod of obtaining an antibody, or binding fragment thereof, that bindsto human NOGO epitope VLPDIVMEAPLN (SEQ ID NO. 60), comprisingimmunising a mammal with said peptide and isolating cells capable ofproducing an antibody which binds to said peptide. In another embodimentof the present invention there is provided a method of obtaining anisolated antibody, or binding fragment thereof, that binds to human NOGOepitope VLPDIVMEAPLN (SEQ ID NO. 60) comprising screening a librarywhich comprises a plurality of antibodies or binding fragments thereof,each being isolatable from the library together with a nucleotidesequence that encodes the antibody or binding fragment thereof, by thebinding of the antibody, or binding fragment thereof to the NOGO epitopeVLPDIVMEAPLN (SEQ ID NO. 60).

Pharmaceutical Compositions

A further aspect of the invention provides a pharmaceutical compositioncomprising an anti-NOGO antibody of the present invention or functionalfragment or equivalent thereof together with a pharmaceuticallyacceptable diluent or carrier.

In a further aspect, the present invention provides a method oftreatment or prophylaxis of stroke (particularly ischemic stroke) andother neurological diseases, in particular Alzheimer's disease, andtreatment of a patient suffering from a mechanical trauma to the CNS(such as spinal chord injury), in a human which comprises administeringto said human in need thereof an effective amount of an anti-NOGOantibody of the invention or functional fragments thereof.

In another aspect, the invention provides the use of an anti-NOGOantibody of the invention or a functional fragment thereof in thepreparation of a medicament for treatment or prophylaxis of stroke(particularly ischemic stroke) and other neurological diseases, inparticular Alzheimer's disease and treatment of a patient suffering froma mechanical trauma to the CNS (such as spinal chord injury).

In a further aspect, the present invention provides a method ofinhibiting neurodegeneration and/or promoting functional recovery in ahuman patient afflicted with, or at risk of developing, a stroke(particularly ischemic stroke) or other neurological disease, inparticular Alzheimer's disease, and treatment of a patient sufferingfrom a mechanical trauma to the CNS (such as spinal chord injury), whichcomprises administering to said human in need thereof an effectiveamount of an anti-NOGO antibody of the invention or a functionalfragment thereof.

In a yet further aspect, the invention provides the use of an anti-NOGOantibody of the invention or a functional fragment thereof in thepreparation of a medicament for inhibiting neurodegeneration and/orpromoting functional recovery in a human patient afflicted with, or atrisk of developing, a stroke and other neurological disease, inparticular Alzheimer's disease and treatment of a patient suffering froma mechanical trauma to the CNS (such as spinal chord injury).

Other aspects and advantages of the present invention are describedfurther in the detailed description and the preferred embodimentsthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: GST-human NOGO-A56 coated at 1.0 mcg/ml. ELISA on purifiedantibodies.

FIG. 2: GST-human NOGO-A56 coated at 0.05 mcg/ml. ELISA on purifiedantibodies.

FIG. 3: GST-human NOGO-A56 coated at 1.0 mcg/ml. ELISA on purifiedantibodies.

FIG. 4: GST-human NOGO-A56 coated at 0.05 mcg/ml. ELISA on purifiedantibodies.

FIG. 5: Epitope mapping using 2A10.

FIG. 6: epitope mapping using H28L16

FIG. 7: Comparison of the binding activity of Hc(G95M)Lc and HcLc asdetermined using a human NOGO-A binding ELISA when NOGO was coated ontoNunc immunosorp plates at 0.05 μg/ml.

FIG. 8: Comparison of the binding activity of Hc(G95M)Lc and HcLc asdetermined using a human NOGO-A binding ELISA when NOGO was coated ontoNunc immunosorp plates at 1 μg/ml.

FIG. 9: Comparison of the binding activity of variants of H6FL incomparison to H6FL L13FL.

FIG. 10: Comparison of the binding activity of variants of H6FL incomparison to H6FL L13FL.

FIG. 11: Direct binding ELISA of the pre-candidate pool antibodies torecombinant human Nogo-A (GST-Nogo-A 5+6). Recombinant GST-Nogo-A 5+6was coated to the plates at A) 1.0 mcg/ml and B) 0.05 mcg/ml. Binding ofthe antibodies was detected using an anti-human IgG-HRP conjugate(Sigma, #A7340X). The negative control was an anti β-amyloid antibody(H2L1). EC50 values were derived using Robosage. Each of the graphsbelow show a representative figure from three independent assays.

FIG. 12: Reverse format binding ELISA of the pre-candidate poolantibodies to recombinant human Nogo-A (GST-Nogo-A 5+6). The anti-Nogo-Aantibodies were captured with anti-human IgG (Sigma, #19764). Thebinding of recombinant GST-Nogo-A 5+6 was detected using an anti-GST-HRPconjugate (Sigma, #A7340). The negative control was an irrelevantantibody. EC50 values were derived using Robosage. The graph below showsa representative figure from three independent assays.

FIG. 13: Competition ELISA of the pre-candidate pool antibodies with theparental antibody 2A10. Recombinant human Nogo-A (GST-Nogo-A 5+6) wascoated to the plates. 2A10 and the humanised antibodies pre-mixed andbinding of 2A10 determined using an anti-mouse IgG-HRP conjugate(Dakocytomation, #P0260). The positive control was HcLc. IC50 valueswere derived using Robosage. The graph below shows a representativefigure from three independent assays.

FIG. 14: Binding of the anti-Nogo-A humanised antibodies to cell-surfaceexpressed human Nogo-A. CHO-K1 cells were engineered to express humanNogo-A. The cells were stained in duplicate with 100 mcg/ml of theanti-Nogo-A humanised antibodies followed by a 1:100 dilution of thePE-labelled anti-human IgG secondary (Sigma, #P8047). An irrelevantantibody was included as a negative control. The data shown are arepresentative example of one of the duplicates.

FIG. 15: Binding of the anti-Nogo-A humanised antibodies tointracellular human Nogo-A. IMR32 cells were permeabilised, fixed andstained with 3-90 mcg/ml of the anti-Nogo-A humanised antibodiesfollowed by 30 mcg/ml of the PE-labelled anti-human IgG secondary (SigmaP-9047). An irrelevant antibody was included as a negative control (-veAb). The data shown below is representative of three independentexperiments.

FIG. 16: Comparison of anti-Nogo-A humanised antibodies in the neuriteoutgrowth assay. H28L16, H27L16 and H20L16 were compared to the parentalantibodies (2A10, HcLc), the antibody 11C7 and various controlantibodies (Control IgG and Campath). Increased neurite-outgrowth wasonly seen with anti-Nogo-A antibodies. The effects were dose-dependentand statistically significant.

FIG. 17: Direct binding ELISA of H28L16 to recombinant full-length humanNogo-A splice (GST-Nogo-A-Biocat 113015). Recombinant Nogo-A splice wascoated to the plates at 1.0 mcg/ml. Binding of the antibodies wasdetected using an anti-human IgG-HRP conjugate (Sigma, #A7340X). Thenegative control was an anti β-amyloid antibody. EC50 values werederived using Robosage. The graph below shows a representative figurefrom two independent assays.

FIG. 18: H28L16 shows reduced C1q binding. ELISA plates were coated witha fixed concentration of the purified humanised and control antibodies(1 mcg/ml). Human C1q (Sigma, C0660) was incubated with the antibodiesand bound C1q quantified using an anti-human c1q-HRP conjugate (TheBinding Site, PP020X). The control antibodies are Campath IgG1, CampathIgG4 and Campath IgG1 Fc-.

FIG. 19: Direct binding ELISA of H28L16, HcLc and 11C7 to GST-NOGO-A56from A) rat B) cynomolgus, C) marmoset and D) squirrel monkey. HcLc wasincluded as a reference. An irrelevant antibody was included as anegative control. The graphs below shows a representative figure fromthree independent assays.

FIG. 20: Competition ELISA to compare the binding epitopes of H28L16 and11C7. Recombinant human Nogo-A (GST-Nogo-A 5+6) was coated to theplates. 2A10 and either 11C7 or H28L16 were pre-mixed and binding of2A10 determined using an anti-mouse IgG-HRP conjugate (Dakocytomation,#P0260). IC50 values were derived using Robosage. The graph below showsa representative figure from three independent assays.

FIG. 21: Competition ELISA to compare the binding of NOGO-5+6(GST-Nogo-A 5+6) and peptide fragments to H28L16. The graph below showsa representative figure from two independent assays.

FIG. 22: ELISA data for the G101S/Q37R variant in comparison with H6L13and H27L16

DETAILED DESCRIPTION OF THE INVENTION

The heavy chain variable regions of the invention may be formatted intothe structure of a natural antibody or functional fragment or equivalentthereof. The antibody may therefore comprise the VH regions of theinvention formatted into a full length antibody, a (Fab′)₂ fragment, aFab fragment, or equivalent thereof (such as scFV, bi- tr- ortetra-bodies, Tandabs, etc.), when paired with an appropriate lightchain. The antibody may be an IgG1, IgG2, IgG3, or IgG4; or IgM; IgA,IgE or IgD or a modified variant thereof. The constant domain of theantibody heavy chain may be selected accordingly. The light chainconstant domain may be a kappa or lambda constant domain. Furthermore,the antibody may comprise modifications of all classes e.g. IgG dimers,Fc mutants that no longer bind Fc receptors or mediate C1q binding. Theantibody may also be a chimeric antibody of the type described inWO86/01533 which comprises an antigen binding region and anon-immunoglobulin region.

The constant region is selected according to the functionality required.Normally an IgG1 will demonstrate lytic ability through binding tocomplement and/or will mediate ADCC (antibody dependent cellcytotoxicity). An IgG4 will be preferred if a non-cytotoxic blockingantibody is required. However, IgG4 antibodies can demonstrateinstability in production and therefore it may be more preferable tomodify the generally more stable IgG1. Suggested modifications aredescribed in EP0307434 preferred modifications include at positions 235and 237. The invention therefore provides a lytic or a non-lytic form ofan antibody according to the invention.

In preferred forms therefore the antibody of the invention is a fulllength (i.e. H2L2 tetramer) non-lytic IgG1 antibody having the heavychain variable regions described herein.

In a further aspect, the invention provides polynucleotides encoding theheavy chain variable regions as described herein.

“NOGO” refers to any NOGO polypeptide, including variant forms. Thisincludes, but is not limited to, NOGO-A having 1192 amino acid residues(GenBank accession no. AJ251383); NOGO-B, a splice variant which lacksresidues 186 to 1004 in the putative extracellular domain (GenBankaccession no. AJ251384) and a shorter splice variant, NOGO-C, which alsolacks residues 186 to 1004 and also has smaller, alternative aminoterminal domain (GenBank accession no. AJ251385) (Prinjha et al (2000)supra). All references to “NOGO” herein is understood to include any andall variant forms of NOGO such as NOGO-A and the splice variantsdescribed, unless a specific form is indicated.

“Neutralising” and grammatical variations thereof refers to inhibition,either total or partial, of NOGO function including its binding toneurones and inhibition of neurite growth.

The terms Fv, Fc, Fd, Fab, or F(ab)₂ are used with their standardmeanings (see, e.g., Harlow et al., Antibodies A Laboratory Manual, ColdSpring Harbor Laboratory, (1988)).

A “chimeric antibody” refers to a type of engineered antibody whichcontains a naturally-occurring variable region (light chain and heavychains) derived from a donor antibody in association with light andheavy chain constant regions derived from an acceptor antibody.

A “humanized antibody” refers to a type of engineered antibody havingits CDRs derived from a non-human donor immunoglobulin, the remainingimmunoglobulin-derived parts of the molecule being derived from one (ormore) human immunoglobulin(s). In addition, framework support residuesmay be altered to preserve binding affinity (see, e.g., Queen et al.,Proc. Natl. Acad Sci USA, 86:10029-10032 (1989), Hodgson et al.,Bio/Technology, 9:421 (1991)). A suitable human acceptor antibody may beone selected from a conventional database, e.g., the KABAT® database,Los Alamos database, and Swiss Protein database, by homology to thenucleotide and amino acid sequences of the donor antibody (in this casethe murine donor antibody 2A10). A human antibody characterized by ahomology to the framework regions of the donor antibody (on an aminoacid basis) may be suitable to provide a heavy chain constant regionand/or a heavy chain variable framework region for insertion of thedonor CDRs (see Table 1 for the 2A10 CDRs for insertion into theacceptor framework). A suitable acceptor antibody capable of donatinglight chain constant or variable framework regions may be selected in asimilar manner. It should be noted that the acceptor antibody heavy andlight chains are not required to originate from the same acceptorantibody. The prior art describes several ways of producing suchhumanised antibodies—see for example EP-A-0239400 and EP-A-054951.

The term “donor antibody” refers to a non-human antibody whichcontributes the amino acid sequences of its variable regions, CDRs, orother functional fragments or analogs thereof to the humanised antibody,and thereby provide the humanised antibody with the antigenicspecificity and neutralizing activity characteristic of the donorantibody.

The term “acceptor antibody” refers to an antibody heterologous to thedonor antibody, which provides the amino acid sequences of its heavyand/or light chain framework regions and/or its heavy and/or light chainconstant regions to the humanised antibody. The acceptor antibody may bederived from any mammal provided that it is non-immunogenic in humans.Preferably the acceptor antibody is a human antibody.

Alternatively, humanisation maybe achieved by a process of “veneering”.A statistical analysis of unique human and murine immunoglobulin heavyand light chain variable regions revealed that the precise patterns ofexposed residues are different in human and murine antibodies, and mostindividual surface positions have a strong preference for a small numberof different residues (see Padlan E. A. et al; (1991) Mol. Immunol. 28,489-498 and Pedersen J. T. et al (1994) J. Mol. Biol. 235; 959-973).Therefore it is possible to reduce the immunogenicity of a non-human Fvby replacing exposed residues in its framework regions that differ fromthose usually found in human antibodies. Because protein antigenicitycan be correlated with surface accessibility, replacement of the surfaceresidues may be sufficient to render the mouse variable region“invisible” to the human immune system (see also Mark G. E. et al (1994)in Handbook of Experimental Pharmacology vol. 113: The pharmacology ofmonoclonal Antibodies, Springer-Verlag, pp 105-134). This procedure ofhumanisation is referred to as “veneering” because only the surface ofthe antibody is altered, the supporting residues remain undisturbed. Afurther alternative approach is set out in WO04/006955.

“CDRs” are defined as the complementarity determining region amino acidsequences of an antibody which are the hypervariable regions ofimmunoglobulin heavy and light chains. See, e.g., Kabat et al.,Sequences of Proteins of Immunological Interest, 4th Ed., U.S.Department of Health and Human Services, National Institutes of Health(1987). There are three heavy chain and three light chain CDRs (or CDRregions) in the variable portion of an immunoglobulin. Thus, “CDRs” asused herein refers to all three heavy chain CDRs, or all three lightchain CDRs (or both all heavy and all light chain CDRs, if appropriate).The structure and protein folding of the antibody may mean that otherresidues are considered part of the antigen binding region and would beunderstood to be so by a skilled person. See for example Chothia et al.,(1989) Conformations of immunoglobulin hypervariable regions; Nature342, p 877-883.

A bispecific antibody is an antibody having binding specificities for atleast two different epitopes. Methods of making such antibodies areknown in the art. Traditionally, the recombinant production ofbispecific antibodies is based on the coexpression of two immunoglobulinH chain-L chain pairs, where the two H chains have different bindingspecificities see Millstein et al, Nature 305 537-539 (1983), WO93/08829and Traunecker et al EMBO, 10, 1991, 3655-3659. Because of the randomassortment of H and L chains, a potential mixture of ten differentantibody structures are produced of which only one has the desiredbinding specificity. An alternative approach involves fusing thevariable domains with the desired binding specificities to heavy chainconstant region comprising at least part of the hinge region, CH2 andCH3 regions. It is preferred to have the CH1 region containing the sitenecessary for light chain binding present in at least one of thefusions. DNA encoding these fusions, and if desired the L chain areinserted into separate expression vectors and are then cotransfectedinto a suitable host organism. It is possible though to insert thecoding sequences for two or all three chains into one expression vector.In one preferred approach, the bispecific antibody is composed of a Hchain with a first binding specificity in one arm and a H-L chain pair,providing a second binding specificity in the other arm, see WO94/04690.See also Suresh et al Methods in Enzymology 121, 210, 1986.

In one embodiment of the invention there is provided a bispecifictherapeutic antibody wherein at least one binding specificity of saidantibody binds to human NOGO at the epitope described in SEQ ID NO. 60.In another embodiment of the present invention the bispecific antibodycomprises the heavy chain variable region CDR H3 sequence MQGY (SEQ IDNO. 45). In another embodiment the bispecific antibody comprises thefollowing pairs of heavy and light chain variable regions: H27L16 (SEQID NO.48+SEQ ID NO.14), H28L13 (SEQ ID NO.49+SEQ ID NO.13) or H28L16(SEQ ID NO.49+SEQ ID NO.14).

The antibodies of the present invention may be produced by transfectionof a host cell with an expression vector comprising the coding sequencefor the antibodies of the invention. An expression vector or recombinantplasmid is produced by placing these coding sequences for the antibodyin operative association with conventional regulatory control sequencescapable of controlling the replication and expression in, and/orsecretion from, a host cell. Regulatory sequences include promotersequences, e.g., CMV promoter, and signal sequences, which can bederived from other known antibodies. Similarly, a second expressionvector can be produced having a DNA sequence which encodes acomplementary antibody light or heavy chain. Preferably this secondexpression vector is identical to the first except insofar as the codingsequences and selectable markers are concerned, so to ensure as far aspossible that each polypeptide chain is functionally expressed.Alternatively, the heavy and light chain coding sequences for thealtered antibody may reside on a single vector.

A selected host cell is co-transfected by conventional techniques withboth the first and second vectors (or simply transfected by a singlevector) to create the transfected host cell of the invention comprisingboth the recombinant or synthetic light and heavy chains. Thetransfected cell is then cultured by conventional techniques to producethe engineered antibody of the invention. The antibody which includesthe association of both the recombinant heavy chain and/or light chainis screened from culture by appropriate assay, such as ELISA or RIA.Similar conventional techniques may be employed to construct otheraltered antibodies and molecules.

One useful expression system is a glutamate synthetase system (such assold by Lonza Biologics), particularly where the host cell is CHO or NS0(see below). Polynucleotide encoding the antibody is readily isolatedand sequenced using conventional procedures (e.g. oligonucleotideprobes). Vectors that may be used include plasmid, virus, phage,transposons, minichromsomes of which plasmids are a typical embodiment.Generally such vectors further include a signal sequence, origin ofreplication, one or more marker genes, an enhancer element, a promoterand transcription termination sequences operably linked to the lightand/or heavy chain polynucleotide so as to facilitate expression.Polynucleotide encoding the light and heavy chains may be inserted intoseparate vectors and introduced (e.g. by electroporation) into the samehost cell or, if desired both the heavy chain and light chain can beinserted into the same vector for transfection into the host cell. Thusaccording to one embodiment of the present invention there is provided aprocess of constructing a vector encoding the light and/or heavy chainsof a therapeutic antibody or antigen binding fragment thereof of theinvention, which method comprises inserting into a vector, apolynucleotide encoding either a light chain and/or heavy chain of atherapeutic antibody of the invention.

In another embodiment there is provided a polynucleotide encoding ahumanised heavy chain variable region having the sequence set forth asSEQ. I.D. NO: 47, 48 or 49.

In another embodiment there is provided a polynucleotide encoding ahumanised heavy chain having the sequence set forth as SEQ. I.D. NO: 53,54 or 55.

It will be immediately apparent to those skilled in the art that due tothe redundancy of the genetic code, alternative polynucleotides to thosedisclosed herein are also available that will encode the polypeptides ofthe invention.

Suitable vectors for the cloning and subcloning steps employed in themethods and construction of the compositions of this invention may beselected by one of skill in the art. For example, the conventional pUCseries of cloning vectors may be used. One vector, pUC19, iscommercially available from supply houses, such as Amersham(Buckinghamshire, United Kingdom) or Pharmacia (Uppsala, Sweden).Additionally, any vector which is capable of replicating readily, has anabundance of cloning sites and selectable genes (e.g., antibioticresistance), and is easily manipulated may be used for cloning. Thus,the selection of the cloning vector is not a limiting factor in thisinvention.

Other preferable vector sequences include a poly A signal sequence, suchas from bovine growth hormone (BGH) and the betaglobin promoter sequence(betaglopro). The expression vectors useful herein may be synthesized bytechniques well known to those skilled in this art. Typical selectiongenes encode proteins that (a) confer resistance to antibiotics or othertoxins e.g. ampicillin, neomycin, methotrexate or tetracycline or (b)complement auxiotrophic deficiencies or supply nutrients not availablein the complex media. The selection scheme may involve arresting growthof the host cell. Cells, which have been successfully transformed withthe genes encoding the therapeutic antibody of the present invention,survive due to e.g. drug resistance conferred by the selection marker.Another example is the so-called DHFR selection marker whereintransformants are cultured in the presence of methotrexate. CHO cellsare a particularly useful cell line for the DHFR selection. Methods ofselecting transformed host cells and amplifying the cell copy number ofthe transgene include using the DHFR system see Kaufman R. J. et al J.Mol. Biol. (1982) 159, 601-621, for review, see Werner R G, Noe W, KoppK, Schluter M,” Appropriate mammalian expression systems forbiopharmaceuticals”, Arzneimittel-Forschung. 48(8):870-80, 1998 August Afurther example is the glutamate synthetase expression system (LonzaBiologics). A suitable selection gene for use in yeast is the trp1 gene;see Stinchcomb et al Nature 282, 38, 1979.

The components of such vectors, e.g. replicons, selection genes,enhancers, promoters, signal sequences and the like, may be obtainedfrom commercial or natural sources or synthesized by known proceduresfor use in directing the expression and/or secretion of the product ofthe recombinant DNA in a selected host. Other appropriate expressionvectors of which numerous types are known in the art for mammalian,bacterial, insect, yeast, and fungal expression may also be selected forthis purpose.

The present invention also encompasses a cell line transfected with arecombinant plasmid containing the coding sequences of the antibodies orequivalents of the present invention. Host cells useful for the cloningand other manipulations of these cloning vectors are also conventional.However, most desirably, cells from various strains of E. coli are usedfor replication of the cloning vectors and other steps in theconstruction of altered antibodies of this invention.

Suitable host cells or cell lines for the expression of the antibody ofthe invention are preferably mammalian cells such as NS0, Sp2/0, CHO(e.g. DG44), COS, a fibroblast cell (e.g., 3T3), and myeloma cells, andmore preferably a CHO or a myeloma cell. Human cells may be used, thusenabling the molecule to be modified with human glycosylation patterns.Alternatively, other eukaryotic cell lines may be employed. Theselection of suitable mammalian host cells and methods fortransformation, culture, amplification, screening and product productionand purification are known in the art. See, e.g., Sambrook et al., citedabove.

Bacterial cells may prove useful as host cells suitable for theexpression of the antibodies or fragments thereof (such as recombinantFabs or ScFvs) of the present invention (see, e.g., Plückthun, A.,Immunol. Rev., 130:151-188 (1992)). However, due to the tendency ofproteins expressed in bacterial cells to be in an unfolded or improperlyfolded form or in a non-glycosylated form, any recombinant fragmentproduced in a bacterial cell would have to be screened for retention ofantigen binding ability. If the molecule expressed by the bacterial cellwas produced in a properly folded form, that bacterial cell would be adesirable host. For example, various strains of E. coli used forexpression are well-known as host cells in the field of biotechnology.Various strains of B. subtilis, Streptomyces, other bacilli and the likemay also be employed in this method.

Where desired, strains of yeast cells known to those skilled in the artare also available as host cells, as well as insect cells, e.g.Drosophila and Lepidoptera and viral expression systems. See, e.g.Miller et al., Genetic Engineering, 8:277-298, Plenum Press (1986) andreferences cited therein.

The general methods by which the vectors may be constructed, thetransfection methods required to produce the host cells of theinvention, and culture methods necessary to produce the antibody of theinvention from such host cell are all conventional techniques.Typically, the culture method of the present invention is a serum-freeculture method, usually by culturing cells serum-free in suspension.Likewise, once produced, the antibodies of the invention may be purifiedfrom the cell culture contents according to standard procedures of theart, including ammonium sulfate precipitation, affinity columns, columnchromatography, gel electrophoresis and the like. Such techniques arewithin the skill of the art and do not limit this invention. Forexample, preparation of antibodies are described in WO 99/58679 and WO96/16990.

Yet another method of expression of the antibodies may utilizeexpression in a transgenic animal, such as described in U.S. Pat. No.4,873,316. This relates to an expression system using the animal'scasein promoter which when transgenically incorporated into a mammalpermits the female to produce the desired recombinant protein in itsmilk.

In a further aspect of the invention there is provided a method ofproducing an antibody of the invention which method comprises the stepof culturing a host cell transformed or transfected with a vectorencoding the light and/or heavy chain of the antibody of the inventionand recovering the antibody thereby produced.

Suitable host cells for cloning or expressing vectors encodingantibodies of the invention are prokaroytic, yeast or higher eukaryoticcells. Suitable prokaryotic cells include eubacteria e.g.enterobacteriaceae such as Escherichia e.g. E. Coli (for example ATCC31,446; 31,537; 27,325), Enterobacter, Erwinia, Klebsiella Proteus,Salmonella e.g. Salmonella typhimurium, Serratia e.g. Serratiamarcescans and Shigella as well as Bacilli such as B. subtilis and B.licheniformis (see DD 266 710), Pseudomonas such as P. aeruginosa andStreptomyces. Of the yeast host cells, Saccharomyces cerevisiae,schizosaccharomyces pombe, Kluyveromyces (e.g. ATCC 16,045; 12,424;24178; 56,500), yarrowia (EP402, 226), Pichia Pastoris (EP183, 070, seealso Peng et al J. Biotechnol. 108 (2004) 185-192), Candida, Trichodermareesia (EP244, 234), Penicillin, Tolypocladium and Aspergillus hostssuch as A. nidulans and A. niger are also contemplated.

Although Prokaryotic and yeast host cells are specifically contemplatedby the invention, typically however, host cells of the present inventionare vertebrate cells. Suitable vertebrate host cells include mammaliancells such as COS-1 (ATCC No.CRL 1650) COS-7 (ATCC CRL 1651), humanembryonic kidney line 293, baby hamster kidney cells (BHK) (ATCCCRL.1632), BHK570 (ATCC NO: CRL 10314), 293 (ATCC NO.CRL 1573), Chinesehamster ovary cells CHO (e.g. CHO-K1, ATCC NO: CCL 61, DHFR-CHO cellline such as DG44 (see Urlaub et al, (1986) Somatic Cell Mol. Genet. 12,555-556)), particularly those CHO cell lines adapted for suspensionculture, mouse sertoli cells, monkey kidney cells, African green monkeykidney cells (ATCC CRL-1587), HELA cells, canine kidney cells (ATCC CCL34), human lung cells (ATCC CCL 75), Hep G2 and myeloma or lymphomacells e.g. NS0 (see U.S. Pat. No. 5,807,715), Sp2/0, Y0.

Thus in one embodiment of the invention there is provided a stablytransformed host cell comprising a vector encoding a heavy chain and/orlight chain of the therapeutic antibody or antigen binding fragmentthereof as described herein. Typically such host cells comprise a firstvector encoding the light chain and a second vector encoding said heavychain.

Host cells transformed with vectors encoding the therapeutic antibodiesof the invention or antigen binding fragments thereof may be cultured byany method known to those skilled in the art. Host cells may be culturedin spinner flasks, roller bottles or hollow fibre systems but it ispreferred for large scale production that stirred tank reactors are usedparticularly for suspension cultures. Typically the stirred tankers areadapted for aeration using e.g. spargers, baffles or low shearimpellers. For bubble columns and airlift reactors direct aeration withair or oxygen bubbles maybe used. Where the host cells are cultured in aserum free culture media it is preferred that the media is supplementedwith a cell protective agent such as pluronic F-68 to help prevent celldamage as a result of the aeration process. Depending on the host cellcharacteristics, either microcarriers maybe used as growth substratesfor anchorage dependent cell lines or the cells maybe adapted tosuspension culture (which is typical). The culturing of host cells,particularly vertebrate host cells may utilise a variety of operationalmodes such as fed-batch, repeated batch processing (see Drapeau et al(1994) cytotechnology 15: 103-109), extended batch process or perfusionculture. Although recombinantly transformed mammalian host cells may becultured in serum-containing media such media comprising fetal calfserum (FCS), it is preferred that such host cells are cultured insynthetic serum-free media such as disclosed in Keen et al (1995)Cytotechnology 17:153-163, or commercially available media such asProCHO-CDM or UltraCHO™ (Cambrex N.J., USA), supplemented wherenecessary with an energy source such as glucose and synthetic growthfactors such as recombinant insulin. The serum-free culturing of hostcells may require that those cells are adapted to grow in serum freeconditions. One adaptation approach is to culture such host cells inserum containing media and repeatedly exchange 80% of the culture mediumfor the serum-free media so that the host cells learn to adapt in serumfree conditions (see e.g. Scharfenberg K et al (1995) in Animal Celltechnology: Developments towards the 21st century (Beuvery E. C. et aleds), pp 619-623, Kluwer Academic publishers).

Antibodies of the invention secreted into the media may be recovered andpurified from the media using a variety of techniques to provide adegree of purification suitable for the intended use. For example theuse of therapeutic antibodies of the invention for the treatment ofhuman patients typically mandates at least 95% purity, more typically98% or 99% purity compared to the culture media comprising thetherapeutic antibodies. In the first instance, cell debris from theculture media is typically removed using centrifugation followed by aclarification step of the supernatant using e.g. microfiltration,ultrafiltration and/or depth filtration. A variety of other techniquessuch as dialysis and gel electrophoresis and chromatographic techniquessuch as hydroxyapatite (HA), affinity chromatography (optionallyinvolving an affinity tagging system such as polyhistidine) and/orhydrophobic interaction chromatography (HIC, see U.S. Pat. No.5,429,746) are available. In one embodiment, the antibodies of theinvention, following various clarification steps, are captured usingProtein A or G affinity chromatography followed by furtherchromatography steps such as ion exchange and/or HA chromatography,anion or cation exchange, size exclusion chromatography and ammoniumsulphate precipitation. Typically, various virus removal steps are alsoemployed (e.g. nanofiltration using e.g. a DV-20 filter). Followingthese various steps, a purified (typically monoclonal) preparationcomprising at least 75 mg/ml or greater e.g. 100 mg/ml or greater of theantibody of the invention or antigen binding fragment thereof isprovided and therefore forms an embodiment of the invention. Suitablysuch preparations are substantially free of aggregated forms ofantibodies of the invention.

In accordance with the present invention there is provided a method ofproducing an anti-NOGO antibody of the present invention whichspecifically binds to and neutralises the activity of human NOGO-A whichmethod comprises the steps of;

-   -   (a) providing a first vector encoding a heavy chain of the        antibody;    -   (b) providing a second vector encoding the light chain of the        antibody;    -   (c) transforming a mammalian host cell (e.g. CHO) with said        first and second vectors;    -   (d) culturing the host cell of step (c) under conditions        conducive to the secretion of the antibody from said host cell        into said culture media;    -   (e) recovering the secreted antibody of step (d).

Once expressed by the desired method, the antibody is then examined forin vitro activity by use of an appropriate assay. Presently conventionalELISA assay formats are employed to assess qualitative and quantitativebinding of the antibody to NOGO. Additionally, other in vitro assays mayalso be used to verify neutralizing efficacy prior to subsequent humanclinical studies performed to evaluate the persistence of the antibodyin the body despite the usual clearance mechanisms.

Other modifications to the antibodies of the present invention includeglycosylation variants of the antibodies of the invention. Glycosylationof antibodies at conserved positions in their constant regions is knownto have a profound effect on antibody function, particularly effectorfunctioning such as those described above, see for example, Boyd et al(1996), Mol. Immunol. 32, 1311-1318. Glycosylation variants of thetherapeutic antibodies or antigen binding fragments thereof of thepresent invention wherein one or more carbonhydrate moiety is added,substituted, deleted or modified are contemplated. Introduction of anasparagine-X-serine or asparagine-X-threonine motif creates a potentialsite for enzymatic attachment of carbonhydrate moieties and maytherefore be used to manipulate the glycosylation of an antibody. InRaju et al (2001) Biochemistry 40, 8868-8876 the terminal sialyation ofa TNFR-IgG immunoadhesin was increased through a process ofregalactosylation and/or resialylation usingbeta-1,4-galactosyltransferace and/or alpha, 2,3 sialyltransferase.Increasing the terminal sialylation is believed to increase thehalf-life of the immunoglobulin. Antibodies, in common with mostglycoproteins, are typically produced in nature as a mixture ofglycoforms. This mixture is particularly apparent when antibodies areproduced in eukaryotic, particularly mammalian cells. A variety ofmethods have been developed to manufacture defined glycoforms, see Zhanget al Science (2004), 303, 371, Sears et al, Science, (2001) 291, 2344,Wacker et al (2002) Science, 298 1790, Davis et al (2002) Chem. Rev.102, 579, Hang et al (2001) Acc. Chem. Res 34, 727. Thus the inventionconcerns a plurality of therapeutic (typically monoclonal) antibodies(which maybe of the IgG isotype, e.g. IgG1) as described hereincomprising a defined number (e.g. 7 or less, for example 5 or less suchas two or a single) glycoform(s) of said antibodies or antigen bindingfragments thereof.

The therapeutic agents of this invention may be administered as aprophylactic or following the stroke event/on-set of clinical symptoms,or as otherwise needed. The dose and duration of treatment relates tothe relative duration of the molecules of the present invention in thehuman circulation, and can be adjusted by one of skill in the artdepending upon the condition being treated and the general health of thepatient. It is envisaged that repeated dosing (e.g. once a week or onceevery two weeks) over an extended time period (e.g. four to six months)maybe required to achieve maximal therapeutic efficacy.

The mode of administration of the therapeutic agent of the invention maybe any suitable route which delivers the agent to the host. Theantibodies, and pharmaceutical compositions of the invention areparticularly useful for parenteral administration, i.e., subcutaneously(s.c.), intrathecally, intraperitoneally (i.p.), intramuscularly (i.m.),intravenously (i.v.), or intranasally (i.n.).

Therapeutic agents of the invention may be prepared as pharmaceuticalcompositions containing an effective amount of the antibody of theinvention as an active ingredient in a pharmaceutically acceptablecarrier. In the prophylactic agent of the invention, an aqueoussuspension or solution containing the engineered antibody, preferablybuffered at physiological pH, in a form ready for injection ispreferred. The compositions for parenteral administration will commonlycomprise a solution of the antibody of the invention or a cocktailthereof dissolved in a pharmaceutically acceptable carrier, preferablyan aqueous carrier. A variety of aqueous carriers may be employed, e.g.,0.9% saline, 0.3% glycine, and the like. These solutions are sterile andgenerally free of particulate matter. These solutions may be sterilizedby conventional, well known sterilization techniques (e.g., filtration).The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, etc. The concentration of theantibody of the invention in such pharmaceutical formulation can varywidely, i.e., from less than about 0.5%, usually at or at least about 1%to as much as 15 or 20% by weight and will be selected primarily basedon fluid volumes, viscosities, etc., according to the particular mode ofadministration selected.

Thus, a pharmaceutical composition of the invention for intramuscularinjection could be prepared to contain 1 mL sterile buffered water, andbetween about 1 ng to about 100 mg, e.g. about 50 ng to about 30 mg ormore preferably, about 5 mg to about 25 mg, of an antibody of theinvention. Similarly, a pharmaceutical composition of the invention forintravenous infusion could be made up to contain about 250 ml of sterileRinger's solution, and about 1 to about 30 and preferably 5 mg to about25 mg of an engineered antibody of the invention per ml of Ringer'ssolution. Actual methods for preparing parenterally administrablecompositions are well known or will be apparent to those skilled in theart and are described in more detail in, for example, Remington'sPharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa.For the preparation of intravenously administrable antibody formulationsof the invention see Lasmar U and Parkins D “The formulation ofBiopharmaceutical products”, Pharma. Sci. Tech. today, page 129-137,Vol. 3 (3^(rd) April 2000), Wang, W “Instability, stabilisation andformulation of liquid protein pharmaceuticals”, Int. J. Pharm 185 (1999)129-188, Stability of Protein Pharmaceuticals Part A and B ed Ahern T.J., Manning M. C., New York, N.Y.: Plenum Press (1992), Akers, M. J.“Excipient-Drug interactions in Parenteral Formulations”, J. Pharm Sci91 (2002) 2283-2300, Imamura, K et al “Effects of types of sugar onstabilization of Protein in the dried state”, J Pharm Sci 92 (2003)266-274, Izutsu, Kkojima, S. “Excipient crystallinity and itsprotein-structure-stabilizing effect during freeze-drying”, J Pharm.Pharmacol, 54 (2002) 1033-1039, Johnson, R, “Mannitol-sucrosemixtures-versatile formulations for protein lyophilization”, J. Pharm.Sci, 91 (2002) 914-922.

Ha, E Wang W, Wang Y. j. “Peroxide formation in polysorbate 80 andprotein stability”, J. Pharm Sci, 91, 2252-2264, (2002) the entirecontents of which are incorporated herein by reference and to which thereader is specifically referred.

It is preferred that the therapeutic agent of the invention, when in apharmaceutical preparation, be present in unit dose forms. Theappropriate therapeutically effective dose will be determined readily bythose of skill in the art. To effectively treat stroke and otherneurological diseases in a human, one dose within the range of 700 to3500 mg per 70 kg body weight of an antibody of this invention isenvisaged to be administered parenterally, preferably s.c., i.v. or i.m.(intramuscularly). Such dose may, if necessary, be repeated atappropriate time intervals selected as appropriate by a physician.

The antibodies described herein can be lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immunoglobulins andart-known lyophilization and reconstitution techniques can be employed.

In another aspect, the invention provides a pharmaceutical compositioncomprising anti-NOGO antibody of the present invention or a functionalfragment thereof and a pharmaceutically acceptable carrier for treatmentor prophylaxis of stroke and other neurological diseases.

In a yet further aspect, the invention provides a pharmaceuticalcomposition comprising the anti-NOGO antibody of the present inventionor a functional fragment thereof and a pharmaceutically acceptablecarrier for inhibiting neurodegeneration and/or promoting functionalrecovery in a human patient suffering, or at risk of developing, astroke or other neurological disease.

The invention further provides a method of treatment or prophylaxis ofstroke (particularly ischemic stroke) and other neurologicaldiseases/disorders, in particular Alzheimer's disease, in a human whichcomprises administering to said human in need thereof an effectiveamount of an anti-NOGO antibody of the present invention or a functionalfragment thereof. Antibodies of the invention may be used in methods oftreatment to slow or halt the progression and/or onset of Alzheimer'sdisease in addition to (or as an alternative to) treating establisheddisease in a human patient.

Further the invention provides the use of an anti-NOGO antibody of thepresent invention, or a functional fragment thereof, in the preparationof a medicament for treatment or prophylaxis of stroke and otherneurological diseases/disorders, in particular Alzheimer's disease.

The invention also provides a method of inhibiting neurodegenerationand/or promoting functional recovery in a human patient suffering, or atrisk of developing, a stroke or other neurological disease/disorder, inparticular Alzheimer's disease, which comprises administering to saidhuman in need thereof an effective amount of an anti-NOGO antibody ofthe present invention or a functional fragment thereof.

In addition the invention provides the use of an anti-NOGO antibody ofthe present invention or a functional fragment thereof in thepreparation of a medicament for inhibiting neurodegeneration and/orpromoting functional recovery in a human patient afflicted with, or atrisk of developing, a stroke and other neurological disease/disorder, inparticular Alzheimer's disease.

The invention further provides a method of treating or prophylaxis ofstroke or other neurological disease/disorder, in particular Alzheimer'sdisease, in a human comprising the step of parenteral administration ofa therapeutically effective amount of an anti-NOGO antibody of thepresent invention. Preferably the said anti-NOGO antibody isadministered intravenously.

Neurological diseases or disorders as used hereinabove includes, but isnot limited to traumatic brain injury, spinal cord injury,fronto-temporal dementias (tauopathies), peripheral neuropathy,Parkinson's disease, Huntington's disease, and in particular Alzheimer'sdisease, multiple sclerosis or amyotrophic lateral sclerosis (ALS).

The invention also provides a method of promoting axonal sproutingcomprising the step of contacting a human axon with an anti-NOGOantibody of the present invention. This method may be performed in-vitroor in-vivo, preferably the method is performed in-vivo.

In a further aspect therefore there is provided a method of treatingstroke (particularly ischemic stroke), brain injury, spinal cord injury,fronto-temporal dementias (tauopathies), peripheral neuropathy,Parkinson's disease, Huntington's disease, multiple sclerosis and inparticular Alzheimer's disease in a human patient which method comprisesthe intravenous administration of a therapeutically effective amount ofan anti-NOGO antibody of the invention.

In a further aspect of the present invention there is provided a methodof promoting axon sprouting of neurons within the central nervous systemof a human subject (e.g. patient) which method comprises administering(e.g. intravenously administering) a therapeutically effective amount ofan anti-NOGO antibody of the present invention.

In a further aspect of the present invention there is provided the useof an anti-NOGO antibody of the present invention (e.g. an anti-NOGOantibody comprising the CDRs set forth herein) in the manufacture of anintravenously administrable medicament for the treatment of stroke(particularly ischemic stroke), brain injury, spinal cord injury,fronto-temporal dementias (tauopathies), peripheral neuropathy,Parkinson's disease, Huntington's disease, and in particular Alzheimer'sdisease, multiple sclerosis or amyotrophic lateral sclerosis (ALS) in ahuman patient.

In a further aspect of the invention there is provided a method ofregenerating axon processes in neurons of the central nervous system ina human patient afflicted with (or susceptible to) stroke (particularlyischemic stroke), brain injury, spinal cord injury, fronto-temporaldementias (tauopathies), peripheral neuropathy, Parkinson's disease,Huntington's disease, multiple sclerosis and in particular Alzheimer'sdisease which method comprises the step of administering (e.g.intravenously) a therapeutically effective amount of an anti-NOGOantibody of the present invention.

In a further aspect of the invention there is provided the use of ananti-NOGO antibody of the present invention in the manufacture of anintravenously administrable pharmaceutical composition for regeneratingaxon processes in neurons of the central nervous system in a humanpatient afflicted with (or susceptible to) stroke (particularly ischemicstroke), brain injury, spinal cord injury, fronto-temporal dementias(tauopathies), peripheral neuropathy, Parkinson's disease, Huntington'sdisease, multiple sclerosis and in particular Alzheimer's disease.

In a further aspect of the invention there is provided a method ofmodulating the production of an amyloidogenic peptide comprisingcontacting a cell which is expressing the precursor from which theamyloidogenic peptide is derived and a NOGO polypeptide (e.g. humanNOGO-A) with an anti-NOGO antibody of the present invention. In typicalembodiments, the precursor is APP. In further typical embodiments theamyloidogenic peptide is Aβ, most preferably Aβ40, Aβ42 or a combinationof both.

As used herein, the term “functional recovery” refers to a motor and/orsensory and/or behavioural improvement in a subject following e.g. anischemic event or injury or on-set of clinical symptoms. Functionalrecovery in humans may be evaluated by instruments designed to measureelemental neurological functions such as motor strength, sensation andcoordination, cognitive functions such as memory, language and theability to follow directions, and functional capacities such as basicactivities of daily living or instrumental activities. Recovery ofelemental neurological function can be measured with instruments such asthe NIH Stroke Scale (NIHSS), recovery of cognitive function can bemeasured with neuropsychological tests such as Boston Naming Test,Trail-making Tests, and California Verbal Learning Test, and activitiesof daily living may be measured with instruments such as the ADCS/ADL(Alzheimer's Disease Clinical Studies/Activities of Daily Living) scaleor the Bristol Activities of Daily Living Scale, all tests and scalesknown in the art.

The following examples illustrate but do not limit the invention.

Example 1 Construction and Expression of Humanised Anti-NOGO Antibodies

Humanised V_(H) and V_(L) constructs were prepared de novo by build upof overlapping oligonucleotides including restriction sites for cloninginto Rld and Rln mammalian expression vectors (or any other suitableexpression vector for expression of proteins in mammalian cells) as wellas a human signal sequence. Hind III and Spe I restriction sites wereintroduced to frame the V_(H) domain containing the CAMPATH-1H signalsequence for cloning into Rld containing the human γ1 mutated constantregion to prevent ADCC and CDC activity (L235A and G237A—EU Indexnumbering system). Hind III and BsiWI restriction sites were introducedto frame the V_(L) domain containing the CAMPATH-1H signal sequence forcloning into Rln containing the human kappa constant region.

CAMPATH-1H signal sequence: (SEQ. ID. NO: 31) MGWSCIILFLVATATGVHS

Plasmids encoding human IgG heavy chain amino acid sequences, whereinthe CDR were that described in table 2, were produced. Plasmids encodinghuman IgG heavy chain amino acid sequences, wherein the CDRs were thatdescribed in table 3, were produced from those existing earlier plasmidsby introducing

single point mutations, G95M (Kabat numbering), using the Quickchangekit (Stratagene).

The following table discloses which full length heavy chain proteinsequences were made in the plasmid vectors and which of the sequenceswere paired, in the sense that the only difference in the amino acidsequences of the paired full length (FL) heavy chain sequences was asubstitution at G95M (kabat numbering) within the CDR H3 of the variableregion:

TABLE 8 G95M substitution CDRs as defined in Table 2 (to form CDR H3 ofTable 3) H1 FL (SEQ ID NO. 35) Not done H6 FL (SEQ ID NO. 15) H26 FL(SEQ ID NO. 53) H16 FL (SEQ ID NO. 16) H27 FL (SEQ ID NO. 54) H20 FL(SEQ ID NO. 42) H28 FL (SEQ ID NO. 55)

Plasmids encoding the heavy chains were then co-transfected into CHOcells (for details see example 2) with the one of the following fulllength light chain sequences: L11 FL (SEQ ID NO. 36), L13 FL (SEQ ID NO.17), or L16 FL (SEQ ID NO. 18).

In parallel a chimera termed HcLc (which is the chimera of 2A10 (SEQ IDNO. 9 and 10—the full length chains comprising the 2A10 murine VH (SEQID NO. 7) and VL (SEQ ID NO.8) and human IgG constant regions)) wasproduced.

Example 2 Antibody Expression in CHO Cells

Rld and Rln plasmids (or other vectors suitable for use in mammaliancells) encoding the heavy and light chains respectively were transientlyco-transfected into CHO cells and expressed at small scale or largescale to produce antibody. Alternatively the same plasmids wereco-transfected into DHFR-CHO cells by electroporation and a stablepolyclonal population of cells expressing the appropriate antibody wereselected using a nucleoside-free media (Rld contains the DHFR gene, Rlncontains a neomycin selection marker). In some assays, antibodies wereassessed directly from the tissue culture supernatant. In other assays,recombinant antibody was recovered and purified by affinitychromatography on Protein A sepharose.

Example 3 Humanised Anti-NOGO Antibody Binds to NOGO

GST-human NOGO-A56 (see example 5) at 0.05-1 μg/ml in PBS was coatedonto Nunc Immunosorp plates (100 μl per well) at 4° C. overnight. Wellswere rinsed once with TBS+0.05% Tween (TBST) then incubated with 2% BSAin TBST to block non-specific binding sites at room temperature for 1hour. Antibodies were diluted in TBST+2% BSA to 10 μg/ml and ½ dilutionsmade from this. Antibodies were added to wells in duplicate andincubated at room temperature for 1 hour. Wells were washed three timeswith TBST then incubated with anti-human kappa peroxidase conjugate(1:2000) for 1 hour. The wells were washed three times with TBST andthen incubated with 100 μl OPD peroxidase substrate (Sigma) per well for10 minutes. The colour reaction was stopped by the addition of 25 μlconcentrated H₂SO₄. Optical density at 490 nm was measured using a platereader. Background values read from wells with no antibody weresubtracted.

FIGS. 1-4 illustrate the dose-dependent binding of humanised antibodiesin comparison with the chimera (termed HcLc which is the chimera of 2A10(comprising the 2A10 murine VH (SEQ ID NO. 7) and VL (SEQ ID NO.8) andhuman IgG constant regions)) to GST-human NOGO-A56 (see Example 5 fordetails) in an ELISA assay. The Y-axis shows the measured opticaldensity (OD) at 490 nm, a quantitative measure of antibody captured inthe wells. The X-axis shows the concentration of antibody used (mcg/ml)per well at each data point.

The antibody material used in FIGS. 1-4 is purified antibody generatedby either the polyclonal expression system or large scale transienttransfections. In these cases, IgG levels were quantified by ELISA andoptical density.

The results from the experiments shown in FIGS. 1-4 shows that theinclusion of the G95M mutation improves the performance of the antibody.The only exception is H27L16 shown in FIGS. 3 and 4 which performed verypoorly. We believe that this data resulted from an unidentifiedtechnical problem with the H27L16 assay, since H27L16 has otherwiseconsistently performed well in other assays (in ELISA shown in FIGS. 1and 2, and in BIAcore assays (Tables 9 and 10)). H27L16 has also beenshown to work very well in later experiments (see FIGS. 11 and 12).

Example 4 Antibody Quantification Protocol

Nunc Immunosorp plates were coated with a goat anti-human IgG chaincapture antibody (Sigma #13382) at 2 μg/ml in Bicarbonate buffer (Sigma#C3041) and incubated overnight at 4° C. The plates were washed twicewith TBS containing 0.05% Tween20 (TBST) and blocked with 200 μl TBSTcontaining 2% (or from 1-3%) BSA (block buffer) for 1 hr at roomtemperature. The plates were washed twice with TBST. Tissue culturesupernatants containing antibody were titrated across the plate in2-fold dilution steps into block buffer and incubated at roomtemperature for 1 hr. The plates were washed three times with TBST. HRPconjugated antibody H23 (goat anti-human kappa chain, Sigma #A7164) wasdiluted 1:2000 in TBST and 100 μl added to each well. The plates wereincubated at room temperature for 1 hr. The plates were washed threetimes with TBST and developed with 100 μl of Fast-OPD substrate (Sigma#P9187). Colour was allowed to develop for 5-10 mins after which timethe ELISA was stopped with 25 μl 3M H₂SO₄. The absorbance at 490 nM wasread plate and antibody concentration determined by reference to astandard curve.

Example 5 Production of NOGO-A Fragment (NOGO-A56, SEQ. ID. NO:32)

A cDNA sequence encoding a polypeptide comprising amino acids 586-785and a GST tag (SEQ. I.D. NO:32) of human NOGO-A was created by cloning acDNA encoding amino acids 586-785 of human NOGO-A into the BamHI-XhoIsites of pGEX-6P1 to generate a GST-tagged fusion protein designatedGST-human-NOGO-A56. Plasmid was expressed in BL21 cells in 2XTY mediumwith 100 μg/ml ampicillin following induction with IPTG to 0.5 mM at 37C for 3 hours. Cell pellets were lysed by sonication and the fusionprotein purified using Glutathione-sepharose (Amersham Pharmacia)following manufacturers instructions. Purified protein was eluted usingreduced glutathione and extensively dialysed against PBS, quantitatedusing BSA standards and a BioRad coomassie based protein assay and thenstored in aliquots at −80 C.

Example 6 BiaCore Analysis of Humanised Anti NOGO Monoclonal Antibodies

The binding kinetics of the anti-NOGO monoclonal antibody (mAb) torecombinantly expressed GST-human NOGO-A was analysed using theBiacore3000 biosensor or BIAcore T100. The hNOGO-A chip was prepared asfollows:

Method

GST-human NOGO-A56 was immobilised to a CM5 chip by primary aminecoupling using the Biacore Wizard program designed for targetedimmobilisation levels. The CM5 sensor surface was activated by passing asolution of 50 mM N-hydroxy-succinimide (NHS) and 200 mMN-ethyl-N′-dimethylaminopropyl carbonide (EDC). Then GST-human NOGO-A56in sodium acetate buffer, pH5.0 or pH 4.5, was passed over the chip andimmobilised. After immobilisation was complete any still activatedesters were blocked by an injection of 1M ethanolamine hydrochloride,pH8.5.

The anti-NOGO mAbs were diluted down in HBS-EP (10 mM HEPES, pH 7.4, 150mM NaCl, 3 mM EDTA, and 0.005% P-20 surfactant) for the BIAcore 3000 orHBS-EP+ (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% P-20surfactant) in the case of the T100 and binding studies were carried outat range of defined antibody concentrations. All runs were referencedagainst a blanked sensor surface (one that had been activated andblocked as described earlier but had no addition of ligand). Analysis ofbinding was carried out using the BIAevaluation kinetic analysissoftware version 4.1 for the BIAcore 3000 and T100 kinetic analysissoftware version 1.0. Biacore analysis of other antibodies of theinvention essentially followed the same protocol as described herein.Unless otherwise stated, the BIAcore experiments were performed at 25°C.

In the following Results section each data table represents the resultsobtained from an individual experiment.

TABLE 9 Results Antibody ka (1/Ms) kd (1/s) KD (pM) HcLc 3.19E6 2.49E−3779 H27L13  6.2E6  1.8E−3 291 H26L13 3.23E6 3.11E−3 963 H28L13 7.26E6 3.3E−3 454 H27L16 6.24E6 1.21E−3 194 H28L16 7.25E6 2.14E−3 296

TABLE 10 Results Antibody ka (1/Ms) kd (1/s) KD (nM) HcLc (25° C.)2.66E6 3.13E−3 1.18 HcLc (37° C.) 5.08E6 7.74E−3 1.46 H16L16 (25° C.)3.43E6 3.72E−3 1.08 H16L16 (37° C.) 5.31E6 6.16E−3 1.16 H20L16 (25° C.)4.69E6 5.42E−3 1.16 H20L16 (37° C.) 7.17E6 1.08E−3 1.51 H27L16 (25° C.)3.94E6 1.50E−3 0.380 H27L16 (37° C.) 7.18E6 3.06E−3 0.426 H27L13 (25°C.) 3.50E6 2.13E−3 0.606 H27L13 (37° C.) 6.58E6 4.22E−3 0.641 H28L16(25° C.) 4.33E6 2.64E−3 0.610 H28L16 (37° C.) 7.73E6 5.24E−3 0.678H28L13 (25° C.) 4.16E6 3.89E−3 0.936 H28L13 (37° C.) 7.43E6 7.59E−3 1.02

TABLE 11 Results Antibodies ka (1/Ms) Kd (1/s) KD (nM) HcLc 3.17E62.33E−3 0.74 H26L13 3.45E6 2.88E−3 0.87 H27L13 6.58E6 1.83E−3 0.28H28L13 6.97E6 3.17E−3 0.45 H28L16 6.89E6 1.95E−3 0.28

Example 7 BiaCore Analysis of Humanised Anti NOGO Monoclonal AntibodiesUsing Off-Rate Ranking

The GST-human NOGO-A56 chip was prepared as for kinetic analysis. Cellsupernatants where taken directly from transient transfections of CHO-K1cells. These were passed directly over the sensor surface and theinteraction measured. A mock transfected cell supernatant was used fordouble referencing to remove any artefacts due to the tissue culturemedia. All runs were referenced against a blanked sensor surface (onethat had been activated and blocked as described earlier but had noaddition of ligand). Analysis of binding was carried out using theBIAevaluation kinetic analysis software version 4.1.

Example 8 Peptide Mapping

47 overlapping peptides spanning the NOGO-A56 portion of GST-humanNOGO-A56 domain (SEQ ID NO. 32) were obtained (from Mimotope™). Thepeptides are 16 amino acids in length with a twelve amino acid overlapwith the adjacent peptide (each peptide further comprising a biotin-SGSGsequence at the N-terminus) with the exception of the first peptidewhich has a GSG-biocytin tag at the C-terminus. The peptides were usedto epitope map the binding site of 2A10 and H28L16.

Method for Epitope Mapping:

Streptavidin at 5 μg/ml in sterile water was coated onto Nunc immunosorpplates (100 μl per well) at 37° C. overnight. The plates were rinsed 3times with PBS containing 0.05% Tween (PBST) then blocked with 3% BSA inPBST at 4° C. overnight. The plates were washed 3 times with PBST.Peptides were then added to the wells at a concentration ofapproximately 10 μg/ml (diluted in 3% BSA in PBST) and incubated at roomtemperature for 1 hour. The plates were washed 3 times with PBST thenincubated for 1 hour with anti-NOGO antibodies diluted to 5 μg/ml in 3%BSA in PBST. The plates were washed 3 times with PBST then incubatedwith anti-human or anti-mouse kappa peroxidase conjugate (1:1000,diluted in 3% BSA in PBST) for 1 hour. The plates were washed 3 timeswith PBST and then incubated with 100 μl OPD peroxidase substrate(Sigma) per well for 10 minutes. The colour reaction was stopped by theaddition of 50 μl 3 molar H₂SO₄. Absorbance at 490 nm was measured usinga plate reader.

The results are shown in FIG. 5 (epitope mapping using H28L16), FIG. 6(epitope mapping using 2A10). FIGS. 5 and 6 show the results of theepitope mapping of 2A10 and H28L16, respectively. The data shownindicates that 2A10 and H28L16 bind to peptides 6 and 7 of which theNOGO specific portion is given in SEQ ID NO.73 and SEQ ID NO.74respectively, both of which contain the sequence VLPDIVMEAPLN (SEQ IDNO.60). These results indicate that VLPDIVMEAPLN (SEQ ID NO.60) containsthe binding epitope of 2A10 and H28L16.

Example 9 Comparison of HcLc and HcLc Containing the G95M Mutation ofthe CDR H3

A modified variant of HcLc was constructed from existing expressionplasmids by introducing a single point mutation, G95M (Kabat numbering),using the Quikchange kit (Stratagene). The protein sequence of thevariable heavy domain Hc (G95M) protein is given in SEQ ID 59.

Hc(G95M)Lc was expressed in CHO cells as described previously. Theantibody was quantified as described in Example 4. FIG. 7 and FIG. 8show a comparison of the binding activity of Hc(G95M)Lc and HcLc asdetermined using a human NOGO-A binding ELISA when NOGO was coated ontoNunc immunosorp plates at 0.05 (FIG. 7) and 1 μg/ml (FIG. 8). Tablebelow shows a comparison of the binding affinities of Hc(G95M)Lc andHcLc.

TABLE 12 Off-rate measured by Biacore ranking based on one experimentSequence ID of heavy Antibody chain variable region Off-rate kd (1/s)H6L13 11 1.38E−2 H6(G95M)L13 47 4.31E−3 HcLc 7 2.66E−3 Hc(G95M)Lc 596.14E−4

The data demonstrates that the G95M substitution within CDR H3 not onlyincreases the binding activity of the humanised antibodies (H6L13), butalso the murine donor antibody 2A10 (HcLc).

Example 10 Construction and Testing of NOGO Antibodies ContainingSubstitutions in CDR H3

A panel of 90 heavy chain variable regions was created by single pointmutations in the residues contained in the CDR H3, or the precedingLeucine. Specifically, vectors encoding a heavy chain (based on H6FL,SEQ ID NO. 15) were made encoding heavy chain variable regions whereeach amino acid residue in CDR H3 and the preceding Leucine wassubstituted (using the Quikchange kit (Stratagene)) with all othernaturally occurring amino acids, excluding cysteine, and expressed inconjunction with a light chain (L13FL, SEQ ID NO. 17) to give 90different antibodies. These antibodies were assayed for binding to NOGOin ELISA and Biacore experiments.

FIGS. 9 and 10 show a comparison of the binding activity of the variantsof H6FL in comparison to H6FL L13FL. Tables 14 and 15 show a comparisonof the off-rate kinetics as measured by Biacore—only the results forthose antibodies that had a measurable off rate in the Biacore assay andhad comparable binding activity to H6L13 in ELISA are shown.

TABLE 14 Parent Antibody VH CDR3 kd (1/s) H6L13 MQGY (SEQ ID NO: 45)4.85E−03 HcLc GQGY (SEQ ID NO: 1) 5.58E−03 H6L13 GQNY (SEQ ID NO: 80)9.66E−03 H6L13 GQLY (SEQ ID NO: 82) 1.32E−02 H6L13 IQGY (SEQ ID NO: 76)1.72E−02 H6L13 RQGY (SEQ ID NO: 75) 1.75E−02 H6L13 GQSY (SEQ ID NO: 62)1.86E−02 H6L13 GQGY (SEQ ID NO: 1) 1.98E−02 H6L13 GSGY (SEQ ID NO: 79)2.07E−02 H6L13 GDGY (SEQ ID NO: 77) 2.12E−02 H6L13 GQGW (SEQ ID NO: 84)2.16E−02 H6L13 GIGY (SEQ ID NO: 78) 2.57E−02 H6L13 GQYY (SEQ ID NO: 81)3.28E−02 H6L13 GQFY (SEQ ID NO: 83) 3.35E−02 H6L13 WQGY (SEQ ID NO: 86)1.98E−02 H6L13 GAGY (SEQ ID NO: 87) 3.15E−02 H6L13 GLGY (SEQ ID NO: 88)1.90E−02 H6L13 GVGY (SEQ ID NO: 89) 1.78E−02 H6L13 GQWY (SEQ ID NO: 90)1.77E−02

Conclusions

The results indicate that the antibodies which retain the bindingproperties of the murine 2A10, and the GQGY containing antibody H6L13,are those containing following CDR H3: RQGY (SEQ ID NO:75), IQGY (SEQ IDNO:76), MQGY (SEQ ID NO:45), GDGY (SEQ ID NO:77), GIGY (SEQ ID NO:78),GSGY (SEQ ID NO:79), GQNY (SEQ ID NO:80), GQYY (SEQ ID NO:81), GQSY (SEQID NO:62), GQLY (SEQ ID NO:82), GQFY (SEQ ID NO:83), GQGW (SEQ IDNO:84), WQGY (SEQ ID NO:86), GAGY (SEQ ID NO:87), GLGY (SEQ ID NO:88),GVGY (SEQ ID NO:89), GQWY (SEQ ID NO:90).

Example 11 Comparison of GQGY Containing Mab (H20L16) with G95M VariantMabs (H27L16 and H28L13 and H28L16)

The antibodies listed in Table 15 were manufactured as described above.

TABLE 15 humanised 2A10 anti-Nogo-A antibodies giving the total numberof back- mutations for the whole antibody (2x heavy chain + 2x lightchain). Antibody Total number of back-mutations per wholeantibody/tetramer H20L16 22 H28L16 22 H28L13 16 H27L16 32

In Vitro Binding Characteristics

In an attempt to rank the antibodies, their binding properties wereinvestigated in a range of assays including ELISA, reverse format ELISA,competition ELISA, Biacore and by flow cytometry.

11.1 Binding to Recombinant Human NOGO-A in ELISA

The ability of the antibodies to bind recombinant human Nogo-A(GST-human Nogo-A 56) was investigated by various related ELISA assays(performed in a related, but slightly different, protocol as thatdescribed in Example 3). In the first assay, the recombinant Nogo-A isdirectly coated to the plate at various different antigenconcentrations. The results of the direct binding ELISA when the antigenis loaded at 1 mcg/ml or 0.05 mcg/ml are shown in FIG. 11A and FIG. 11Brespectively. The data confirms that all the antibodies show comparablebinding activity to recombinant human Nogo-A when compared with thechimeric form of the parental antibody (HcLc). At higher antigen coatingconcentrations, all antibodies yield a similar EC50 value. In contrast,at a lower antigen coating concentration the assay was able todiscriminate between the antibodies. Although saturation curves were notobtained, a trend analysis on the lines revealed the following rankorder: H27L16>H28L16, H28L13, H20L16.

In a parallel experiment, the format of the assay was reversed. In thisformat, the antibody is captured on to the plate and the binding of therecombinant human Nogo-A (GST-human Nogo-A-56) detected using the GSTtag. The results of the reverse format ELISA are shown in FIG. 12. Thedata confirms that all the antibodies show comparable binding activityto recombinant human Nogo-A when compared with the chimeric form of theparental antibody (HcLc). This format of the binding ELISA did notdistinguish between the antibodies.

11.2 Competition ELISA

The ability of the antibodies to compete directly with the parentalantibody for the same epitope on human Nogo-A was assessed using acompetition ELISA. The recombinant human Nogo-A (GST-human Nogo-A 56)was coated onto the plates. The parental antibody 2A10 and the humanisedantibodies were pre-mixed prior to adding to the plates. The binding of2A10 was quantified using an anti-mouse IgG-HRP conjugate(Dakocytomation, #P0260). The results shown in FIG. 13 confirm that allfour antibodies can compete with 2A10. This suggests that the humanisedantibodies and parental antibody recognise an overlapping epitope onhuman Nogo-A. Furthermore, the activity of the humanised antibodies iscomparable or better than the chimera HcLc. The results indicate thatH27L16, H28L16 and H28L13 are more potent than H20L16.

11.3 Biacore Affinity Measurements

Biacore was used to determine affinities and rank antibodies using twodifferent methodologies. In the first approach, the recombinant Nogo-Awas coupled to the surface of the chip and anti-Nogo-A antibodies passedover this surface. In the second approach, Protein A was used to capturethe antibody onto the surface of the chip over which the recombinantGST-human Nogo-A56 was passed. The results shown in Table 16 wereobtained by coupling the antigen to the surface and confirm that allfour antibodies show comparable/better affinity than the parentalantibody (HcLc). Based on the average of six independent runs, theantibodies rank in the following order in terms of overall affinity:H27L16>H28L16>H28L13>H20L16, consistent with the rank order of thedirect binding ELISA (FIG. 11B). In the case of H27L16 and H28L16, thehumanised antibodies demonstrate 2-3× higher affinity that the parentalantibody (HcLc).

TABLE 16 Binding kinetics of the anti-Nogo-A humanised antibodies torecombinant human Nogo-A (GST-human Nogo-A 56) as determined using theBiacore T100. The antigen was bound to the CM5 chip by primary aminecoupling. The antibodies were flowed over a various concentrations(0.125-8 nM). The values show the mean and standard deviation (inbrackets) of six independent runs carried out in duplicate. Eachcompleted data set was analysed independently prior to the calculationof mean and standard deviation. Antibody Ka kd KD (nM) H20L16** 5.37E6(7.65E5) 9.70E−3 (2.65E−3) 1.80 (0.31) H27L16 3.96E6 (9.93E5) 2.30E−3(1.11E−3) 0.56 (0.15) H28L13 8.13E6 (1.35E6) 9.10E−3 (2.65E−3) 1.11(0.18) H28L16 6.97E6 (6.62E5) 4.43E−3 (1.18E−3) 0.64 (0.15) HcLc 3.80E6(7.11E5) 7.09E−3 (2.22E−3) 1.86 (0.32) **Only 11 sets of data analysedfor H20L16 as one set could not be analysed.

In a similar manner to the ELISA, the kinetics of antibody binding torecombinant human Nogo-A (GST-human Nogo-A 56) was also assessed in areverse format (see Example 11.1). In this assay, the humanisedantibodies were captured onto the CM5 chip by Protein A. The averagedresults for six independent runs are shown in Table 17. Consistent withthe reverse format ELISA, all the humanised Nogo-A antibodies showsimilar binding kinetics to the chimera (HcLc) in the reverse formatBiacore.

TABLE 17 Reverse format binding kinetics of the anti-Nogo-A humanisedantibodies to recombinant human Nogo-A (GST Nogo-A 5 + 6) as determinedusing the Biacore T100. Protein A was immobilised to approximately4000RUs by primary amine and used to capture 200-300RUs of the sampleantibodies. Recombinant human Nogo-A was passed over at variousconcentrations (0.125-8 nM). The values show the mean and standarddeviation (in brackets) of three independent runs in duplicate. Eachdata set was independently analysed prior to the calculation of the meanand standard deviation. Antibody Ka kd KD (nM) H20L16 1.01E6 (1.35E5)3.13E−4 (2.79E−5) 0.31 (0.036) H27L16 9.93E5 (2.02E4) 3.04E−4 (1.83E−5)0.31 (0.019) H28L13 1.12E6 (1.21E5) 3.84E−4 (3.24E−5) 0.34 (0.015)H28L16 1.18E6 (8.32E4) 4.01E−4 (2.48E−5) 0.34 (0.032) HcLc 1.38E6(3.70E5) 5.69E−4 (1.54E−4) 0.41 (0.062)

11.4 Binding to Native Human NOGO

To demonstrate that the humanised antibodies bind to native human Nogo-Awith a profile comparable to the parental antibody, two flow cytometrybased assays were developed. In the first assay, a CHO-K1-based cellline expressing human Nogo-A extracellular domain on the cell surfacewas generated. Binding of the humanised anti-Nogo-A antibodies wasassessed by flow cytometry using a PE-labelled anti-human IgG (Sigma,#P8047). FIG. 14 below shows a typical profile for the anti-Nogo-Aantibodies on the CHO-Nogo-A cell line. Whilst the assay is notsensitive enough to distinguish between the antibodies, the resultsconfirm that all four antibodies can recognise cell surface expressedhuman Nogo-A at levels comparable to that of the chimera. None of theantibodies recognise the parental cell line (CHO-K1—data not shown).

In the second assay, the ability of the humanised antibodies to bindnative Nogo-A was assessed using a human neuroblastoma cell line—IMR32.This cell line is characterised by high intracellular/low cell surfacelevels of Nogo-A protein. In an attempt to increase the binding signal,the assay was set-up to detect intracellular Nogo-A (ER-resident). IMR32cells were permeabilised and fixed prior to staining with theanti-Nogo-A humanised antibodies. Binding of the antibodies to Nogo-Awas detected using an anti-human IgG-PE labelled secondary (Sigma,#P8047). The results, shown in FIG. 15 below, confirm that all theantibodies bind to intracellular Nogo-A at levels comparable or higherthan the parental antibody HcLc. These data, in conjunction with theresults from the CHO-Nogo-A cell line, confirm that the humanisedantibodies can recognise a more native form of the Nogo-A protein atlevels comparable or better than the chimera, HcLc. The assays are notsufficiently sensitive to rank the antibody panel.

11.5 Neurite-Outgrowth Assays

Humanised anti-Nogo-A antibodies were tested for their ability toneutralise neurite-outgrowth (NO) inhibitory activity of Nogo-A in anassay that is based on quantifying NO as described previously.Antibodies tested in the assay were selected on the basis of theirbinding kinetics for Nogo-A. High affinity humanised antibodies namely,H28L16, H27L16, H20L16 and for reference their parental antibodies 2A10(mouse monoclonal) and HcLc (human mouse chimera) were tested for Nogo-Aneutralisation. For comparison, antibody 11C7 (see Example 13) was alsotested in the assay.

In order to test the neutralising activity of selected humanisedantibodies, wells coated with human recombinant GST-human Nogo-A56 andtreated with varying concentrations of antibodies at 37° C. for 1 hprior to the addition of cerebellum granular neurons (CGNs). Controlwells were treated with HBSS. Average neurite length per neurite wasmeasured for each well. FIG. 16 shows the results for the humanisedantibodies tested in the assay. A panel of control antibodies (controlIgG, purified mouse IgG; Campath and another irrelevant humanisedantibodies) used to confirm the specificity of the activity. As afurther control, the same humanised antibodies were titrated onto GSTcoated plates. The results confirm that H28L16, H27L16 and H20L16reverse Nogo-A-mediated inhibition of neurite outgrowth to a similardegree observed for the parental antibodies (2A10 and HcLc). The effectsappear to be robust and stable and were seen with H28L16 in eight out ofeleven independent neurite-outgrowth experiments. In contrast, thehumanised antibodies do not increase neurite-outgrowth on GST coatedplates and the panel of control antibodies do not show any dosedependent reversal of inhibition, confirming that the effect of thehumanised antibodies is specific for Nogo-A-mediated inhibition. Thedata presented for the neurtite outgrowth is selected from number ofrepeat experiments. Whilst a number of the repeats which are not shownappeared to be variable in nature, it is believed that the data shownreflects a true activity of the antibodies of the present invention inreducing the inhibitory effect of NOGO in the neurite outgrowth assay.

Example 12 Further Characterisation of H28L 16 12.1 Binding toFull-Length Recombinant Nogo-A

The ability of the antibodies to bind full-length extracellular domainrecombinant human Nogo-A (GST-human Nogo-A-ECD) was investigated by adirect binding ELISA assay. In this case the ECD was a splice variantfalling within the region of approximately position 186-1004 of humanNOGO A (the portion beginning DETFAL (SEQ ID NO.95) and ending withELSKTS (SEQ ID NO.96)).

The recombinant GST-human Nogo-A-ECD was directly coated to the plate at1 μg/ml. The data shown in FIG. 17 confirms that H28L16 can recogniseGST-human Nogo-A-ECD as levels comparable or better than the parental(HcLc) or H20L16.

12.2 Inhibition of Fc Functionality

To improve the safety profile of the candidate, residues L235 and G237within the CH2 domain of the heavy chain constant region (EU Indexsystem) were mutated to alanine residues thus reducing the likelihood oftriggering antibody-mediated immunological effector functions. Reducedhuman C1q binding was used as a surrogate for inhibition of Fcfunctionality. FIG. 18 below shows that H28L16 has significantly reducedC1q binding activity, compared to Campath-IgG1 (wild-type) andcomparable to a Campath IgG1 construct bearing the same mutations(Fc-mutated antibody (Fc-)) and Campath IgG4. These data suggest thatthe CH2-domain mutations present in H28L16 will significantly reduce thelikelihood of triggering Fc mediated effector functions.

12.3 Orthologue Binding

To confirm that H28L16 shows binding activity to various orthologues ofNogo-A, comparable to that of the parental antibody (HcLc), a series ofbinding assays were performed. FIG. 19 A-D below shows the results of adirect binding ELISA to recombinant NOGO (GST-human Nogo-A 56) from rat(SEQ ID NO.94), cynomolgus (SEQ ID NO. 92), marmoset (SEQ ID NO. 93) andsquirrel monkey respectively (SEQ ID NO. 91). In all cases, H28L16 showsactivity comparable or better than the chimeric antibody (HcLc). Thecalculated EC50 values are very similar to those calculated for bindingto human recombinant Nogo-A.

The kinetics of binding of H28L16 to the various orthologues of Nogo-Ain comparison to HcLc and 11C7 was determined using the Biacore. Table18 and Table 19 below show the kinetics of binding in two differentformats of the assay. Where the recombinant Nogo-A was coupled directlyto the CM5 chip (Table 18), the binding kinetics for rat, cynomolgusmonkey, squirrel monkey and marmoset are very similar to that for human(range=0.33-0.67 nM). When the format of the assay was reversed and theantibodies are captured onto the chip using Protein A (Table 19), thebinding affinity of H28L16 to rat Nogo-A is approximately 4-fold lowerthan for human Nogo-A. A similar trend is observed for cynomolgus Nogo-A(8.5× lower affinity than human) and the other primate orthologues(12-17× lower affinity than human). The chimeric antibody HcLc shows asimilar profile of binding to the orthologues of Nogo-A in bothorientations of the assay. Since it is unclear which assay format bestrepresents the in vivo situation, the primary conclusions that can bedrawn from this study are 1) H28L16 has retained the orthologuecross-reactivity profile associated with the chimeric antibody HcLc and2) the affinity of HcLc for rat and cynomolgus Nogo-A is within 4-foldand 8.5-fold of the affinity for human Nogo-A and under certainconditions may be very similar.

TABLE 18 Binding kinetics of H28L16, 11C7 and HcLc to the recombinantorthologues of human Nogo-A as determined using the Biacore T100.Approximately 140-180RUs of the various Nogo-A orthologues were capturedto the CM5 chip by primary amine coupling. The antibodies were flowedover a various concentrations (0.125-8 nM). The values show the mean andstandard deviation (in brackets) of 1-2 independent runs carried out induplicate with each data set independently analysed prior to calculationof the mean and standard deviation. H28L16 11C7 HcLc Orthologue Ka Kd KD(nM) Ka Kd KD (nM) Ka Kd KD (nM) Cynomolgus 4.65E6 3.07E−3 0.67 1.47E63.40E−4 0.23 2.94E6 4.78E−3 1.68 (2 runs)* (7.47E5) (2.37E−4) (0.06)(1.67E5) (4.45E−5) (0.01) (7.13E5) (6.34E−4) (0.35) Rat 4.64E6 1.54E−30.33 8.36E5 1.20E−4 0.11 2.53E6 2.83E−3 1.12 (2 runs) (2.34E5) (3.06E−5)(0.01) (5.58E5) (2.14E−5) (0.03) (5.32E4) (2.30E−5) (0.03) Marmoset 4.2E6 3.02E−3 0.626 1.16E6 2.80E−4 0.24 3.13E6 4.44E−3 1.419 (1 run)(2.47E4) (5.09E−5) (0.000) (5.37E4) (6.15E−6) (0.006) (2.76E4) (1.41E−4)(0.03) Squirrel 4.46E6 2.73E−3 0.61 1.10E6 2.86E−4 0.26 3.04E6 4.68E−31.54 Monkey (6.08E4) (4.95E−6) (0.000) (3.25E4) (1.87E−5) (0.010)(1.64E5) (2.11E−4) (0.15) (1 run) Human 6.97E6 4.43E−3 0.64 1.58E62.64E−4 0.19 3.80E6 7.09E−3 1.86 (6.62E5) (1.18E−3) (0.15) (6.42E5)(5.57E−5) (7.96E−2) (7.11E5) (2.22E−3) (0.32) *One set of curves wasdiscarded due to uninterpretable curves for antibody 11C7.

TABLE 19 Reverse format binding kinetics of H28L16, 11C7 and HcLc to therecombinant orthologues of human Nogo-A as determined on the BiacoreT100. Protein A was immobilised on the surface at about 4000RUs andanti-Nogo-A antibodies were captured at approximately 300-400RUs. Therecombinant proteins (GST-NOGO-A56) were flowed over a variousconcentrations (0.125-64 nM) dependent on the construct. All the runswere done in duplicates. The values show the mean and standard deviation(in brackets) of 1-3 independent runs with each run done in duplicateand each data set analysed independently prior to calculation of themean and standard deviation. H28L16 11C7 HcLc KD KD KD Orthologue Ka Kd(nM) Ka Kd (nM) Ka Kd (nM) Cynomolgus 3.26E5 1.11E−3 3.41 4.02E5 2.97E−40.76 3.03E5 1.41E−3 4.66 (3 runs) (4.06E3) (2.23E−5) (0.05) (6.85E4)(1.11E−5) (0.12) (4.58E3) (2.84E−5) (0.08) Rat 3.80E5 6.69E−4 1.762.83E5 1.77E−4 0.64 5.47E5 1.10E−3 2.01 (3 runs) (5.68E3) (1.24E−5)(0.03) (4.66E4) (1.34E−5) (0.09) (1.20E4) (2.86E−5) (0.07) Marmoset2.22E5 1.09E−3 4.89 1.91E5 2.54E−4 1.33 3.02E5 1.36E−3 4.51 (1 run)(3.61E3) (7.35E−5) (0.25) (2.90E3) (3.46E−6) (0.00) (9.90E2) (7.92E−5)(0.28) Squirrel 1.57E5 1.08E−3 6.86 1.03E5 2.78E−4 2.69 1.74E5 1.29E−37.45 Monkey (1 (2.69E3) (5.02E−5) (0.20) (2.12E3) (3.61E−6) (0.02)(2.19E3) (7.64E−5) (0.34) run) Human 1.20E6 4.75E−4 0.40 2.64E5 1.49E−40.57 1.32E6 7.00E−4 0.54 (1 run) (8.49E4) (9.97E−6) (0.02) (3.32E3)(1.61E−5) (0.07) (2.71E5) (3.18E−5) (0.09)

12.4 Physical Properties

The physicochemical properties of H28L16 and H20L16 were assessed bySEC-HPLC and SDS-PAGE. SEC-HPLC was carried out at 1.0 ml/minute using100 mM sodium phosphate, 400 mM sodium chloride pH 6.8 and a TSK G3000SW×l30 cm×7.8 mm stainless steel column with detection at 214 nm and 280nm. SDS-PAGE was carried out on a 4-20% Novex Tris-HCL gel loading 10 μgproduct and staining with Sypro Ruby. C-IEF was carried out on a BeckmanMDQ using pH 3.5-10 ampholines. The following results were obtained:

TABLE 20 Size exclusion chromatography (SEC) HPLC analysis of theanti-Nogo-A antibodies. The values shown are percentages of the antibodyassigned to each of the three different species. Antibody Aggregate %Monomer % Fragment % H28L16 0.50 99.50 0.00 H20L16 14.21 85.75 0.05

TABLE 21 SDS-PAGE analysis of the anti-Nogo-A antibodies. The valuesshown are percentages of the antibody found in the major bands. AntibodyNon-reduced Reduced H28L16 82.4% HC: 67.2% LC: 27.7% H + L: 94.9% H20L1684.6% HC: 69.3% LC: 26.4% H + L: 95.7%

The SEC-HPLC data suggests that H20L16 is more susceptible toaggregation than H28L16 (H28L16). If the data reported here were to berepeated at large scale, this could impact the ability of themanufacturing process to produce material of acceptable quality forclinical use (>95% monomer). The SDS-PAGE data shows both candidates areacceptable with both showing a typical profile.

Example 13 Comparison of H28L16 with 11C7

A murine anti-Nogo-A antibody designated 11C7 is described inWO2004052932, which was raised to a peptide epitope. A chimeric 11C7 wasmade based on the sequence information provided in WO2004052932. Tocompare the binding epitopes of 2A10 and 11C7, a competition ELISA wasestablished to investigate if 11C7 and 2A10 recognise an overlappingepitope on Nogo-A. As shown in FIG. 20 below, HcLc (the chimeric form of2A10) was able to compete with 2A10 for binding to human recombinantNogo-A whereas 11C7 showed no competition with 2A10, even atconcentrations of up to 100 mcg/ml.

Example 14 Competition ELISA to Demonstrate the Ability of Peptides toCompete Directly with Human NOGO-5+6 for Binding to NOGO H28L 16 Methodfor Competition ELISA

The ability of peptides to compete directly with NOGO-A (GST-humanNogo-A56) for binding to NOGO H28L16 was assessed using a competitionELISA. Rabbit anti-human IgG (Sigma, #1-9764) at 5 g/ml in bicarbonatebuffer was coated onto Nunc immunosorp plates (100 ul per well) at 4° C.overnight. The plates were rinsed 3 times with TBS containing 0.05%Tween (TBST) then blocked with 1% BSA in TBST at room temperature for 1hour. H28L16 was then captured onto the plate (1 ug/ml, diluted in 1%BSA in TBST, 50 ul per well) at room temperature for 1 hour. The plateswere washed 3 times with TBST. Peptides (from 0 to 100 g/ml) andGST-human NOGO-A56 at a concentration of 1 ug/ml (diluted in 1% BSA inTBST) were pre-mixed prior to addition into the wells and incubated atroom temperature for 1 hour. The plates were washed 3 times with TBSTthen incubated for 1 hour with rabbit anti-GST peroxidase conjugate(Sigma, #A7340, 1:2000, diluted in 1% BSA in TBST) for 1 hour. Theplates were washed 3 times with TBST and then incubated with 50 l OPDperoxidase substrate (Sigma) per well for 10 minutes. The colourreaction was stopped by the addition of 25 l concentrated H₂SO₄.Absorbance at 490 nm was measured using a plate reader.

The results shown in FIG. 21 confirm that peptides 6 and 7, which werepositive in the epitope mapping ELISA (Example 8) can compete withGST-human NOGO-A56 binding to H28L16. This suggests that the peptideswhich were positive in the epitope mapping study contain an epitope forH28L16 binding. Peptides 16 and 17 (which contain NOGO peptides, but notoverlapping with peptides 6 or 7), which do not contain the proposedepitope, do not compete with NOGO-5+6.

Example 15 ELISA Analysis of a Humanised Anti NOGO Monoclonal AntibodyBased on the NOGO antibody variant G101S/Q37R

G101S (also known as H100 (SEQ ID NO.63)), a modified variant of theheavy chain variable region of H6 (SEQ ID NO.11) was generated byintroducing a single substitution, G101S (Kabat numbering) into CDR H3as described above. Similarly, Q37R, a modified variant of the lightchain variable region of L13 (SEQ ID NO. 13) were generated byintroducing a single substitution (Kabat numbering Q37R) into theframework region (to form L100). The protein sequence of the variablelight domain Q37R is given in SEQ ID NO. 67.

Genes encoding full length versions of the heavy and light chainscontaining the G101S/Q37R substitutions were expressed in CHO cells asdescribed previously and assayed in a direct binding ELISA as describedpreviously.

The results of the direct binding ELISA when the antigen is loaded at0.05 ug/ml are shown in FIG. 22. The data confirms that antibodyH100L100 shows comparable binding activity to recombinant GST-humanNOGO-A56 when compared with H27L16 and that H100L100 has an improvedbinding profile when compared to H6L13. Corresponding EC50 values areshown in the table below:

TABLE 22 EC50 measurements for the G101S/Q37R variant in comparison withH6L13 and H27L16 Antibody EC50 value H6L13 0.086 H27L16 0.052 H100/L1000.048

Example 16 BiaCore Analysis of Humanised Anti NOGO Monoclonal AntibodiesBased on the CDR H3 Variant G101S

H100, A modified variant of the heavy chain variable region of H6 (SEQID NO.11) was generated by introducing a single substitution, G101S(Kabat numbering) into CDR H3. The protein sequence of the variableheavy domain H100 protein is given in SEQ ID NO.63. Similarly, L100 andL101, modified variants of the light chain variable region of L13 (SEQID NO. 13) were generated by introducing a single substitution (Kabatnumbering Q37R and Q45R respectively) into the framework region. Theprotein sequences of the variable light domains L100 and L101 proteinsare given in SEQ ID NO.67 and SEQ ID NO.68 respectively.

Full length versions of H100L100 and H100L101 were expressed in CHOcells as described previously. Table 23 shows a comparison of thebinding affinities of H6L13 with H100L100 and H100L101 and indicatesthat H100L100 and H100L101 have an improved binding affinity whencompared with H6L13. In this example, the method was performedessentially as described in Example 6 where the CM5 chip was activatedby passing the NHS and EDC solutions over the chip at 5 μl/ml for 7minutes and the NOGO was suspended in 10 nM sodium acetate buffer (pH4.5) before passing over the chip.

TABLE 23 Biacore measurements for the G101S variants of the H6 variableheavy chain in combination with variants of the L13 variable light chainin comparison with H6L13. Antibody On rate ka (1/Ms) Off-rate kd (1/s)Affinity (KD, nM) H6L13 1.04E+06 7.22E−03 6.97 H100L100 1.28E+075.07E−03 0.396 H100L101 1.30E+07 4.29E−03 0.329

TABLE 24 NOGO antibody sequences Summary Sequence identifier (SEQ. I.D.NO) amino acid Polynucleotide Description sequence sequence 2A10, CDR-H11 — 2A10, CDR-H2 2 — 2A10, CDR-H3 3 — 2A10, CDR-L1 4 — 2A10, CDR-L2 5 —2A10, CDR-L3 6 — 2A10, VH (murine) 7 19 2A10, VL (murine) 8 20 Chimericheavy chain Hc 9 21 Chimeric light chain Lc 10 22 2A10 VH humanisedconstruct H6 11 23 2A10 VH humanised construct H16 12 24 2A10 VLhumanised construct L13 13 25 2A10 VL humanised construct L16 14 26 2A10heavy chain humanised construct H6 15 27 2A10 heavy chain humanisedconstruct 16 28 H16 2A10 light chain humanised construct L13 17 29 2A10light chain humanised construct L16 18 30 Campath leader sequence 31 —Amino acids 586-785 of human NOGO A 32 — (NOGO-A56) fused to GST 2A10 VHhumanised construct H1 33 37 2A10 VL humanised construct L11 34 38 2A10heavy chain humanised construct H1 35 39 2A10 light chain humanisedconstruct L11 36 40 2A10 VH humanised construct H20 41 43 2A10 heavychain humanised construct 42 44 H20 2A10, CDR-H3 (G95M) 45 Sequence ofMarmoset NOGO-A fragment 46 VH humanised construct H26 47 50 VHhumanised construct H27 48 51 VH humanised construct H28 49 52 Heavychain humanised construct H26 53 56 Heavy chain humanised construct H2754 57 Heavy chain humanised construct H28 55 58 Chimeric heavy chain Hc(G95M) 59 Epitope 60 2A10 VH humanised construct H99 61 CDR (G101S) 62VH humanised construct H100 63 VH humanised construct H101 64 VHhumanised construct H102 65 VH humanised construct H98 66 L100 (L13 +Q37R) 67 L101 (L13 + Q45R) 68 L102 (L13 + Q37R/Q45R) 69 L103 (L16 +Q37R) 70 L104 (L16 + Q45R) 71 L105 (L16 + Q37R/Q45R) 72 Peptide 73peptide 74 CDR H3 analogue 75 CDR H3 analogue 76 CDR H3 analogue 77 CDRH3 analogue 78 CDR H3 analogue 79 CDR H3 analogue 80 CDR H3 analogue 81CDR H3 analogue 82 CDR H3 analogue 83 CDR H3 analogue 84 NOGO peptide 85CDR H3 analogue 86 CDR H3 analogue 87 CDR H3 analogue 88 CDR H3 analogue89 CDR H3 analogue 90 Squirrel monkey NOGO (A56) plus GST 91 tagCynomolgus monkey NOGO (A56) plus 92 GST tag Marmoset NOGO (A56) plusGST tag 93 Rat NOGO (A56) plus GST tag 94 Human NOGO peptide 95 HumanNOGO peptide 96

Sequences SEQ ID NO. 1 2A10 CDR-H1 SYWMH SEQ ID NO. 2: 2A10 CDR-H2NINPSNGGTNYNEKFKS SEQ ID NO. 3: 2A10 CDR-H3 GQGY SEQ ID NO. 4:2A10 CDR-L1 RSSKSLLYKDGKTYLN SEQ ID NO. 5: 2A10 CDR-L2 LMSTRASSEQ ID NO. 6: 2A10 CDR-L3 QQLVEYPLT SEQ ID NO. 7: 2A10, VH (murine)QVQLQQPGTELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGNINPSNGGTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCELGQGYWGQGTTLTVSS SEQ ID NO. 8: 2A10, VL (murine)DIVITQDELSNPVTSGESVSISCRSSKSLLYKDGKTYLNWFLQRPGQSPQLLIYLMSTRASGVSDRFSGSGSGTDFTLEISRVKAEDVGVYYCQQLVEYPLTFGAGTKLELK SEQ ID NO. 9:Chimeric heavy chain HcMGWSCIILFLVAAATGVHSQVQLQQPGTELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGNINPSNGGTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCELGQGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO. 10: Chimeric light chain LcMRCSLQFLGVLMFWISGVSGDIVITQDELSNPVTSGESVSISCRSSKSLLYKDGKTYLNWFLQRPGQSPQLLIYLMSTRASGVSDRFSGSGSGTDFTLEISRVKAEDVGVYYCQQLVEYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID 11: 2A10 VH humanised construct H6QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGNINPSNGGTNYNEKFKSRATMTRDTSTSTAYMELSSLRSEDTAVYYCELGQGYWGQGTLVTVSS SEQ ID NO. 122A10 VH humanised construct H16QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVKQRPGQGLEWIGNINPSNGGTNYNEKFKSKATLTVDKSTSTAYMELSSLRSEDTAVYYCELGQGYWGQGTLVTVSS SEQ ID NO. 13:2A10 VL humanised construct L13DIVMTQSPLSLPVTLGQPASISCRSSKSLLYKDGKTYLNWFQQRPGQSPQLLIYLMSTRASGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCQQLVEYPLTFGQGTKLEIK SEQ ID NO. 14:2A10 VL humanised construct L16DIVMTQSPLSNPVTLGQPVSISCRSSKSLLYKDGKTYLNWFLQRPGQSPQLLIYLMSTRASGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCQQLVEYPLTFGQGTKLEIK SEQ ID NO. 15:2A10 heavy chain humanised construct H6MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGNINPSNGGTNYNEKFKSRATMTRDTSTSTAYMELSSLRSEDTAVYYCELGQGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO. 16: 2A10 heavy chain humanised construct H16MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVKQRPGQGLEWIGNINPSNGGTNYNEKFKSKATLTVDKSTSTAYMELSSLRSEDTAVYYCELGQGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO. 17: 2A10 light chain humanised construct L13MGWSCIILFLVATATGVHSDIVMTQSPLSLPVTLGQPASISCRSSKSLLYKDGKTYLNWFQQRPGQSPQLLIYLMSTRASGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCQQLVEYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 18: 2A10 light chain humanised construct L16MGWSCIILFLVATATGVHSDIVMTQSPLSNPVTLGQPVSISCRSSKSLLYKDGKTYLNWFLQRPGQSPQLLIYLMSTRASGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCQQLVEYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 19: PN encoding 2A10, VH (murine) SEQ ID: 7CAGGTCCAACTGCAGCAGCCTGGGACTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAAATATTAATCCTAGCAATGGTGGTACTAACTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTATTGTGAACTGGGACAGGGCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA SEQ ID NO. 20:PN encoding 2A10, VL (murine) SEQ ID: 8GATATTGTGATAACCCAGGATGAACTCTCCAATCCTGTCACTTCTGGAGAATCAGTTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTATATAAGGATGGGAAGACATACTTGAATTGGTTTCTGCAGAGACCAGGACAATCTCCTCAGCTCCTGATCTATTTGATGTCCACCCGTGCATCAGGAGTCTCAGACCGGTTTAGTGGCAGTGGGTCAGGAACAGATTTCACCCTGGAAATCAGTAGAGTGAAGGCTGAGGATGTGGGTGTGTATTACTGTCAACAACTTGTAGAGTATCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA SEQ ID NO. 21:PN encoding Chimeric heavy chain Hc SEQ ID: 9ATGGGATGGAGCTGTATCATCCTCTTTTTGGTAGCAGCAGCTACAGGTGTCCACTCCCAGGTCCAACTGCAGCAGCCTGGGACTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGGAAATATTAATCCTAGCAATGGTGGTACTAACTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTATTGTGAACTGGGACAGGGCTACTGGGGCCAAGGCACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA SEQ ID NO. 22: PN encoding Chimeric light chain Lc SEQ ID: 10ATGAGGTGCTCTCTTCAGTTTCTGGGGGTGCTTATGTTCTGGATCTCTGGAGTCAGTGGGGATATTGTGATAACCCAGGATGAACTCTCCAATCCTGTCACTTCTGGAGAATCAGTTTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTATATAAGGATGGGAAGACATACTTGAATTGGTTTCTGCAGAGACCAGGACAATCTCCTCAGCTCCTGATCTATTTGATGTCCACCCGTGCATCAGGAGTCTCAGACCGGTTTAGTGGCAGTGGGTCAGGAACAGATTTCACCCTGGAAATCAGTAGAGTGAAGGCTGAGGATGTGGGTGTGTATTACTGTCAACAACTTGTAGAGTATCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGACAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG SEQ ID NO. 23:PN encoding 2A10 VH humanised construct H6 SEQ ID: 11CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTGGATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATCGGAAATATTAATCCTAGCAATGGTGGTACTAACTACAATGAGAAGTTCAAGAGCAGAGCCACCATGACCAGGGACACGTCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGAACTGGGACAGGGCTACTGGGGCCAGGGAACACTAGTCACAGTCTCCTCA SEQ ID NO. 24:PN encoding 2A10 VH humanised construct H16 SEQ ID: 12CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTGGATGCACTGGGTGAAACAGCGACCTGGACAAGGGCTTGAGTGGATCGGAAATATTAATCCTAGCAATGGTGGTACTAACTACAATGAGAAGTTCAAGAGCAAAGCCACCCTCACCGTCGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGAACTGGGACAGGGCTACTGGGGCCAGGGAACACTAGTCACAGTCTCCTCA SEQ ID NO. 25:PN encoding 2A10 VL humanised construct L13 SEQ ID: 13GATATTGTGATGACCCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTATATAAGGATGGGAAGACATACTTGAATTGGTTTCAGCAGAGGCCAGGCCAATCTCCACAGCTCCTAATTTATTTGATGTCCACCCGTGCATCTGGGGTCCCAGACAGATTCAGCGGCGGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCCAACAACTTGTAGAGTATCCGCTCACGTTTGGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO. 26:PN encoding 2A10 VL humanised construct L16 SEQ ID: 14GATATTGTGATGACCCAGTCTCCACTCTCCAACCCCGTCACCCTTGGACAGCCGGTCTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTATATAAGGATGGGAAGACATACTTGAATTGGTTTCTCCAGAGGCCAGGCCAATCTCCACAGCTCCTAATTTATTTGATGTCCACCCGTGCATCTGGGGTCCCAGACAGATTCAGCGGCGGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCCAACAACTTGTAGAGTATCCGCTCACGTTTGGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO. 27:PN encoding 2A10 heavy chain humanised construct H6 SEQ ID: 15ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTGGATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATCGGAAATATTAATCCTAGCAATGGTGGTACTAACTACAATGAGAAGTTCAAGAGCAGAGCCACCATGACCAGGGACACGTCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGAACTGGGACAGGGCTACTGGGGCCAGGGAACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA SEQ ID NO. 28:PN encoding 2A10 heavy chain humanised construct H16 SEQ ID: 16ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTGGATGCACTGGGTGAAACAGCGACCTGGACAAGGGCTTGAGTGGATCGGAAATATTAATCCTAGCAATGGTGGTACTAACTACAATGAGAAGTTCAAGAGCAAAGCCACCCTCACCGTCGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGAACTGGGACAGGGCTACTGGGGCCAGGGAACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA SEQ ID NO. 29:PN encoding 2A10 light chain humanised construct L13 SEQ ID: 17ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCGATATTGTGATGACCCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTATATAAGGATGGGAAGACATACTTGAATTGGTTTCAGCAGAGGCCAGGCCAATCTCCACAGCTCCTAATTTATTTGATGTCCACCCGTGCATCTGGGGTCCCAGACAGATTCAGCGGCGGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCCAACAACTTGTAGAGTATCCGCTCACGTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGACAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG SEQ ID NO. 30:PN encoding 2A10 light chain humanised construct L16 SEQ ID: 18ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCGATATTGTGATGACCCAGTCTCCACTCTCCAACCCCGTCACCCTTGGACAGCCGGTCTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTATATAAGGATGGGAAGACATACTTGAATTGGTTTCTCCAGAGGCCAGGCCAATCTCCACAGCTCCTAATTTATTTGATGTCCACCCGTGCATCTGGGGTCCCAGACAGATTCAGCGGCGGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCCAACAACTTGTAGAGTATCCGCTCACGTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGACAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG SEQ ID NO. 31: Campath leader sequenceMGWSCIILFLVATATGVHS SEQ ID NO. 32:Amino acids 586-785 of human NOGO A (NOGO-A56) fused to GSTMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSMQESLYPAAQLCPSFEESEATPSPVLPDIVMEAPLNSAVPSAGASVIQPSSSPLEASSVNYESIKHEPENPPPYEEAMSVSLKKVSGIKEEIKEPENINAALQETEAPYISIACDLIKETKLSAEPAPDFSDYSEMAKVEQPVPDHSELVEDSSPDSEPVDLFSDDSIPDVPQKQDETVMLVKESLTETSFESMIEYENKELERPHRD SEQ ID NO. 33:2A10 VH humanised construct H1QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGNINPSNGGTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCELGQGYWGQGTLVTVSS SEQ ID NO. 34:2A10 VL humanised construct L11DIVITQSPLSLPVTLGQPASISCRSSKSLLYKDGKTYLNWFQQRPGQSPQLLIYLMSTRASGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCQQLVEYPLTFGQGTKLEIK SEQ ID NO. 35:2A10 heavy chain humanised construct H1MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGNINPSNGGTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCELGQGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO. 36: A10 light chain humanised construct L11MGWSCIILFLVATATGVHSDIVITQSPLSLPVTLGQPASISCRSSKSLLYKDGKTYLNWFQQRPGQSPQLLIYLMSTRASGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCQQLVEYPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 37:PN encoding 2A10 VH humanised construct H1 SEQ ID: 33CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTGGATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAATATTAATCCTAGCAATGGTGGTACTAACTACAATGAGAAGTTCAAGAGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGAACTGGGACAGGGCTACTGGGGCCAGGGAACACTAGTCACAGTCTCCTCA SEQ ID NO. 38:PN encoding 2A10 VL humanised construct L11 SEQ ID: 34GATATTGTGATAACCCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTATATAAGGATGGGAAGACATACTTGAATTGGTTTCAGCAGAGGCCAGGCCAATCTCCACAGCTCCTAATTTATTTGATGTCCACCCGTGCATCTGGGGTCCCAGACAGATTCAGCGGCGGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCCAACAACTTGTAGAGTATCCGCTCACGTTTGGCCAGGGGACCAAGCTGGAGATCAAA SEQ ID NO. 39:PN encoding 2A10 humanised heavy chain H1 SEQ ID: 35ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTGGATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGAAATATTAATCCTAGCAATGGTGGTACTAACTACAATGAGAAGTTCAAGAGCAGAGTCACCATGACCAGGGACACGTCCACGAGCACAGTCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGAACTGGGACAGGGCTACTGGGGCCAGGGAACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA SEQ ID NO. 40:PN encoding 2A10 humanised light chain construct L11 SEQ ID: 36ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCGATATTGTGATAACCCAGTCTCCACTCTCCCTGCCCGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTATATAAGGATGGGAAGACATACTTGAATTGGTTTCAGCAGAGGCCAGGCCAATCTCCACAGCTCCTAATTTATTTGATGTCCACCCGTGCATCTGGGGTCCCAGACAGATTCAGCGGCGGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCCAACAACTTGTAGAGTATCCGCTCACGTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGACAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAG SEQ ID NO. 41: 2A10 VH humanised construct H20QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGNINPSNGGTNYNEKFKSKATMTRDTSTSTAYMELSSLRSEDTAVYYCELGQGYWGQGTLVTVSS SEQ ID NO. 42:2A10 heavy chain humanised construct H20MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGNINPSNGGTNYNEKFKSKATMTRDTSTSTAYMELSSLRSEDTAVYYCELGQGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO. 43: PN encoding 2A10 VH humanised construct H20 SEQ ID: 41CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTGGATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATCGGAAATATTAATCCTAGCAATGGTGGTACTAACTACAATGAGAAGTTCAAGAGCAAGGCCACCATGACCAGGGACACGTCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGAACTGGGACAGGGCTACTGGGGCCAGGGAACACTAGTCACAGTCTCCTCA SEQ ID NO. 44:PN encoding 2A10 heavy chain humanised construct H20 SEQ ID: 42ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTGGATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATCGGAAATATTAATCCTAGCAATGGTGGTACTAACTACAATGAGAAGTTCAAGAGCAAGGCCACCATGACCAGGGACACGTCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGAACTGGGACAGGGCTACTGGGGCCAGGGAACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA SEQ ID NO. 45: 2A10 CDR-H3 (G95M) MQGY SEQ ID NO. 46:Amino acid sequence of Marmoset NOGO-A fragmentVQDSLCPVAQLCPSFEESEATPSPVLPDIVMEAPLNSAVPSAGASAVQPSSSPLEASSVNFESVKHEPENPPPYEEAMNVSRKKVSGIKEEIKEPESINAAVQETEAPYISIACDLIKETKLSAEPTPDFSSYSEMAKVEQPLPDHSELVEDSSPDSEPVDLFSDDSIPDVPQKQDEAVILVKETLTETSFESMIEHENK SEQ ID NO. 47:VH humanised construct H26QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGNINPSNGGTNYNEKFKSRATMTRDTSTSTAYMELSSLRSEDTAVYYCELMQGYWGQGTLVTVSS SEQ ID NO. 48:VH humanised construct H27QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVKQRPGQGLEWIGNINPSNGGTNYNEKFKSKATLTVDKSTSTAYMELSSLRSEDTAVYYCELMQGYWGQGTLVTVSS SEQ ID NO. 49:VH humanised construct H28QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGNINPSNGGTNYNEKFKSKATMTRDTSTSTAYMELSSLRSEDTAVYYCELMQGYWGQGTLVTVSS SEQ ID NO. 50:PN encoding VH humanised construct H26 SEQ ID: 47CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTGGATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATCGGAAATATTAATCCTAGCAATGGTGGTACTAACTACAATGAGAAGTTCAAGAGCAGAGCCACCATGACCAGGGACACGTCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGAACTGATGCAGGGCTACTGGGGCCAGGGAACACTAGTCACAGTCTCCTCA SEQ ID NO. 51:PN encoding VH humanised construct H27 SEQ ID: 48CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTGGATGCACTGGGTGAAACAGCGACCTGGACAAGGGCTTGAGTGGATCGGAAATATTAATCCTAGCAATGGTGGTACTAACTACAATGAGAAGTTCAAGAGCAAAGCCACCCTCACCGTCGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGAACTGATGCAGGGCTACTGGGGCCAGGGAACACTAGTCACAGTCTCCTCA SEQ ID NO. 52:PN encoding VH humanised construct H28 SEQ ID: 49CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTGGATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATCGGAAATATTAATCCTAGCAATGGTGGTACTAACTACAATGAGAAGTTCAAGAGCAAGGCCACCATGACCAGGGACACGTCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGAACTGATGCAGGGCTACTGGGGCCAGGGAACACTAGTCACAGTCTCCTCA SEQ ID NO. 53:Heavy chain humanised construct H26MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGNINPSNGGTNYNEKFKSRATMTRDTSTSTAYMELSSLRSEDTAVYYCELMQGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO. 54: Heavy chain humanised construct H27MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVKQRPGQGLEWIGNINPSNGGTNYNEKFKSKATLTVDKSTSTAYMELSSLRSEDTAVYYCELMQGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO. 55: Heavy chain humanised construct H28MGWSCIILFLVATATGVHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGNINPSNGGTNYNEKFKSKATMTRDTSTSTAYMELSSLRSEDTAVYYCELMQGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO. 56:PN encoding Heavy chain humanised construct H26 SEQ ID: 53ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTGGATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATCGGAAATATTAATCCTAGCAATGGTGGTACTAACTACAATGAGAAGTTCAAGAGCAGAGCCACCATGACCAGGGACACGTCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGAACTGATGCAGGGCTACTGGGGCCAGGGAACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA SEQ ID NO. 57:PN encoding Heavy chain humanised construct H27 SEQ ID: 54ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTGGATGCACTGGGTGAAACAGCGACCTGGACAAGGGCTTGAGTGGATCGGAAATATTAATCCTAGCAATGGTGGTACTAACTACAATGAGAAGTTCAAGAGCAAAGCCACCCTCACCGTCGACAAATCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGAACTGATGCAGGGCTACTGGGGCCAGGGAACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA SEQ ID NO. 58:PN encoding Heavy chain humanised construct H28 SEQ ID: 55ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGTGTCCACTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGATACACCTTCACCAGCTACTGGATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATCGGAAATATTAATCCTAGCAATGGTGGTACTAACTACAATGAGAAGTTCAAGAGCAAGGCCACCATGACCAGGGACACGTCCACGAGCACAGCCTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGTGAACTGATGCAGGGCTACTGGGGCCAGGGAACACTAGTCACAGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCGCGGGGGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA SEQ ID NO. 59: Heavy chain Hc (G95M)MGWSCIILFLVAAATGVHSQVQLQQPGTELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGNINPSNGGTNYNEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCELMQGYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID 60: Epitope VLPDIVMEAPLN SEQ ID 61:2A10 VH humanised construct H99QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGNINPSNGGTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCELGQSYWGQGTLVTVSS SEQ ID NO. 62: CDR H3 GQSYSEQ ID NO. 63: VH humanised construct H100QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGNINPSNGGTNYNEKFKSRATMTRDTSTSTAYMELSSLRSEDTAVYYCELGQSYWGQGTLVTVSS SEQ ID NO. 64:VH humanised construct H101QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVKQRPGQGLEWIGNINPSNGGTNYNEKFKSKATLTVDKSTSTAYMELSSLRSEDTAVYYCELGQSYWGQGTLVTVSS SEQ ID NO. 65:VH humanised construct H102QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWIGNINPSNGGTNYNEKFKSKATMTRDTSTSTAYMELSSLRSEDTAVYYCELGQSYWGQGTLVTVSS SEQ ID NO. 662A10 VH humanised construct H98QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQGLEWMGNINPSNGGTNYNEKFKSRVTMTRDTSTSTVYMELSSLRSEDTAVYYCELMQGYWGQGTLVTVSS SEQ ID NO. 67DIVMTQSPLSLPVTLGQPASISCRSSKSLLYKDGKTYLNWFRQRPGQSPQLLIYLMSTRASGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCQQLVEYPLTFGQGTKLEIK SEQ ID NO. 68DIVMTQSPLSLPVTLGQPASISCRSSKSLLYKDGKTYLNWFQQRPGQSPRLLIYLMSTRASGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCQQLVEYPLTFGQGTKLEIK SEQ ID NO. 69DIVMTQSPLSLPVTLGQPASISCRSSKSLLYKDGKTYLNWFRQRPGQSPRLLIYLMSTRASGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCQQLVEYPLTFGQGTKLEIK SEQ ID NO. 70DIVMTQSPLSNPVTLGQPVSISCRSSKSLLYKDGKTYLNWFRQRPGQSPQLLIYLMSTRASGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCQQLVEYPLTFGQGTKLEIK SEQ ID NO. 71DIVMTQSPLSNPVTLGQPVSISCRSSKSLLYKDGKTYLNWFLQRPGQSPPLLIYLMSTRASGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCQQLVEYPLTFGQGTKLEIK SEQ ID NO. 72DIVMTQSPLSNPVTLGQPVSISCRSSKSLLYKDGKTYLNWFRQRPGQSPRLLIYLMSTRASGVPDRFSGGGSGTDFTLKISRVEAEDVGVYYCQQLVEYPLTFGQGTKLEIK SEQ ID NO. 73 TPSPVLPDIVMEAPLNSEQ ID NO. 74 VLPDIVMEAPLNSAVP SEQ ID NO. 75 RQGY SEQ ID NO. 76 IQGYSEQ ID NO. 77 GDGY SEQ ID NO. 78 GIGY SEQ ID NO. 79 GSGY SEQ ID NO. 80GQNY SEQ ID NO. 81 GQYY SEQ ID NO. 82 GQLY SEQ ID NO. 83 GQFYSEQ ID NO. 84 GQGW SEQ ID NO. 85 YESIKHEPENPPPYEE SEQ ID NO. 86 WQGYSEQ ID NO. 87 GAGY SEQ ID NO. 88 GLGY SEQ ID NO. 89 GVGY SEQ ID NO. 90GQWY SEQ ID NO. 91MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSMQESLYPVAQLCPSFEESEATPSPVLPDIVMEAPLNSAVPSAVASAVQPSLSPLEASSVNYESVKHEPENPPPYEEAMNVSLKKVSGIKEEIKEPESIKAAVQETEAPYISIACDLIKETKLSAEPTPDFSNYSEMAKVEQPLPDHSEIVEDSSPDSEPVDLFSDDSIPDVPQKQDEAVILVKENLTETSFESMIEHENKLERPHRD SEQ ID NO. 92MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSKMDLVQTSEVMQESLYPAAQLCPSFEESEATPSPVLPDIVMEAPLNSAVPSAGASAVQPSSSPLEASSVNYESIIHEPENPPPYEEAMSVSLKKVSGIKEEIKEPESINAAVQETEAPYISIACDLIKETKLSAEPTPDFSDYSEMAKVEQPVPDHSELVEDSSPDSEPVDLFSDDSIPDVPQKQDEAVMLVKENLPETSFESMIEHENKEKLSALPPEGGSSGRIVTDSEQ ID NO. 93MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSVQDSLCPVAQLCPSFEESEATPSPVLPDIVMEAPLNSAVPSAGASAVQPSSSPLEASSVNFESVKHEPENPPPYEEAMNVSRKKVSGIKEEIKEPESINAAVQETEAPYISIACDLIKETKLSAEPTPDFSSYSEMAKVEQPLPDHSELVEDSSPDSEPVDLFSDDSIPDVPQKQDEAVILVKETLTETSFESMIEHENKLERPHRD SEQ ID NO. 94MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKSDLEVLFQGPLGSIQESLYPTAQLCPSFEEAEATPSPVLPDIVMEAPLNSLLPSAGASVVQPSVSPLEAPPPVSYDSIKLEPENPPPYEEAMNVALKALGTKEGIKEPESFNAAVQETEAPYISIACDLIKETKLSTEPSPDFSNYSEIAKFEKSVPEHAELVEDSSPESEPVDLFSDDSIPEVPQTQEEAVMLMKESLTEVSETVAQHKEERL SEQ ID NO. 95 DETFALSEQ ID NO. 96 ELSKTS

1. A heavy chain variable region comprising a third CDR consistingessentially of the amino acid residues GQGY wherein the CDR contains atleast one substitution within the GQGY core sequence, the substitutionsbeing selected from the following substitutions: where the G in thefirst position is replaced by R, I, W or M; the Q in the second positionis replaced by D, I, A, L, V or S; the G in the third position isreplaced by W, N, Y, S, L or F; and the Y in the fourth position isreplaced by W.
 2. A heavy chain variable region as claimed in claim 1wherein there is a single substitution to the GQGY sequence to yield oneof the following CDR H3: RQGY (SEQ ID NO.75), IQGY (SEQ ID NO.76), MQGY(SEQ ID NO.45), GDGY (SEQ ID NO.77), GIGY (SEQ ID NO.78), GSGY (SEQ IDNO.79), GQNY (SEQ ID NO.80), GQYY (SEQ ID NO.81), GQSY (SEQ ID NO.62),GQLY (SEQ ID NO.82), GQFY (SEQ ID NO.83), GQGW (SEQ ID NO.84), WQGY (SEQID NO.86), GAGY (SEQ ID NO.87), GLGY (SEQ ID NO.88), GVGY (SEQ IDNO.89), GQWY (SEQ ID NO.90).
 3. A heavy chain variable region as claimedin claim 2 wherein the CDR H3 is MQGY or GQSY.
 4. A heavy chain variableregion as claimed in claim 1, wherein the heavy chain variable regioncomprises the sequence SYWMH as CDR H1 (SEQ ID NO. 1) andNINPSNGGTNYNEKFKS as CDR H2 (SEQ ID NO.2).
 5. A heavy chain variableregion as claimed in claim 1, wherein the heavy chain variable region isa humanised sequence.
 6. A heavy chain variable region as claimed inclaim 5 wherein the acceptor heavy chain variable region sequence has atleast 40% identity in the framework regions to the 2A10 donor antibodyheavy chain variable region sequence given in SEQ ID NO.7.
 7. A heavychain variable region as claimed in claim 6 wherein the heavy chainvariable region has an amino acid sequence of SEQ ID NO. 66 (H98variable region) or SEQ ID NO. 61 (H99 variable region), furthercomprising a number of substitutions at one or more of numericalpositions 38, 40, 67, 68, 70, 72, 74, and 79; wherein each substitutedamino acid residue is replaced with the amino acid residue at theequivalent position in SEQ ID NO
 7. 8. A heavy chain variable region asclaimed in claim 1, having the amino acid sequence given in SEQ ID NO.47(H26), SEQ ID NO.48 (H27), SEQ ID NO.49 (H28), SEQ ID NO. 63 (H100), SEQID NO. 54 (H101), SEQ ID NO. 65 (H102).
 9. An isolated antibody, orfragment thereof, capable of binding to human NOGO-A comprising a heavychain variable region as claimed in claim 1 and a light chain variableregion.
 10. An isolated antibody or fragment thereof as claimed in claim7 comprising the following heavy and light chain variable region pairs:H27L16 (SEQ ID NO.48+SEQ ID NO.14), H28L13 (SEQ ID NO.49+SEQ ID NO.13),H28L16 (SEQ ID NO.49+SEQ ID NO.14).
 11. An isolated antibody as claimedin claim 9 comprising the following heavy and light chain sequencesH27FL L16FL (SEQ ID NO. 54+SEQ ID NO.18), H28FL L13FL (SEQ ID NO. 55+SEQID NO.17), H28FL L16FL (SEQ ID NO. 55+SEQ ID NO.18).
 12. Apharmaceutical composition comprising an anti-NOGO antibody or fragmentthereof comprising the antibodies or fragments thereof as claimed inclaim 9, together with a pharmaceutically acceptable diluent or carrier.13. Use of an anti-NOGO antibody or fragment thereof as claimed in claim9, in the preparation of a medicament for treatment or prophylaxis ofstroke and other neurological diseases/disorders or for the treatment ofa patient suffering from a mechanical trauma to the central orperipheral nervous system.
 14. A method for the treatment or prophylaxisof stroke or other neurological disease/disorder or for the treatment ofa patient suffering from a mechanical trauma to the central orperipheral nervous system, in a human comprising the step of parenteraladministration of a therapeutically effective amount of an anti-NOGOantibody or fragment thereof as claimed in claim
 9. 15. An antibody orfragment thereof that is capable of binding to a region of human NOGOprotein consisting of the polypeptide sequence of VLPDIVMEAPLN (SEQ IDNO. 60), characterised in that the antibody, or fragment thereof is notan antibody comprising a variable heavy domain having CDR H3 consistingof the amino acid residues GQGY or analogues thereof having one aminoacid substitution therein.
 16. (canceled)
 17. An antibody, or fragmentthereof, that is capable of binding to human NOGO protein in an ELISAassay, wherein the binding of the antibody, or fragment thereof, tohuman NOGO protein is reduced in the presence of a peptide having thefollowing sequence VLPDIVMEAPLN (SEQ ID NO. 60), and is not reduced inthe presence of an irrelevant peptide, characterised in that theantibody or fragment thereof is not an antibody comprising a heavy chainvariable domain having a CDR H3 consisting of the amino acid residuesGQGY or analogues thereof having one amino acid substitution therein.